- Season 1: Batman and the others go on a mission to defeat and capture villains. Along the way, they meet Jason Todd a.k.a. The Red Hood. In the end, they now must stop Hugo Strange from killing the patients.
- Season 2: After Arkham City is shut down, a dangerous threat arrives and threatens to destroy humanity. They get help from Batman's Earth 3 counterpart.
- Crisis on Two Earths arc of season 3: On Earth 2, the Justice Underground (Legion of Doom's good counterparts) call upon Batman and the others heroes to save their world from a terrorist organization called the Crime Syndicate (Justice League's evil counterparts).
- The Court of Owls arc of season 3: Owlman is back but this time, he has made a new group called the Court of Owls. Also, it's later revealed that he is Batman's older half-brother Thomas Wayne Jr.
- Circus of Strange arc of season 4: Gordon goes into war with a crime lord named Billy Maroni, Sal Maroni's crazy son but gets help from Sofia Falcone, Carmine's daughter. Meanwhile, Batman goes face-to-face with Mario Calvi, Vandal Savage, and Professor Pyg.
- A Dark Knight arc of season 4: When crime continues, Jason Todd goes on a mission to stop Sofia Falcone and end her empire.
- Kevin Conroy as Bruce Wayne/Batman - One of the Main Protagonist of the show alongside Robin and title character. David Mazouz plays a younger Bruce.
- Michael Cera as Tim Drake/Red Robin - One of the Main Protagonists of the show alongside Batman and Batman's sidekick.
- Mila Kunis as Barbara Gordon/Batgirl
- Andrew Garfield as Richard Grayson/Nightwing
- Jensen Ackles as Jason Todd/Red Hood - A violent member of the Bat Family and sometimes tries to kill criminals. He has a hi-tech red mask.
- Martin Jarvis as Alfred Pennyworth
- Chris Parnell as Commissioner James Gordon
- Kurt Russell as Captain Harvey Bullock
- Michelle Rodriquez as Renee Montoya - A newbie cop and former love-interest of Bruce Wayne. She starts to fall in love with Jason Todd.
- Samuel L. Jackson as Lucius Fox
- Jackie Earle Harley as Hugo Strange - The Main Antagonist of season 1 and warden of Arkham City. He is later killed by both Mr. Freeze and Flame Queen.
- James Frain as Theo Galavan/Azazel - The Secondary Antagonist of season 1 and Hugo's second in command.
- William Fichtner as Jean-Paul Valley/Doom - The Main Antagonist of season 2 and Batman's black lantern counterpart. His costume resembles the Azrael version of the bat-suit. When he is in disguise, he is voiced by Tony Todd. His Earth is called Earth-5 because he is an evil version of Batman and is revealed to be a Black Lantern. His goal is to destroy both humanity and the black lanterns (including his boss Solar) (all failed but managed to betray and kill Zoom).
- Daniel Radcliffe as Connor Kent a.k.a. Superboy - He is the genetic clone son of Superman and Wonder Woman.
- Jared Padalecki as Damian al Ghul/Robin II - A new Robin and member of Young Justice.
- Nicholas D'Agosto as Jamie Hayes a.k.a. Blue Beetle
- Ben Affleck as V-Bat - Batman's Earth 3 counterpart and Secondary Hero in Blackest Night arc of season 2. Unlike Batman, V-Bat kills criminals.
- J.K. Simmons as Earth 3 Jim Gordon
- Patrick Stewart as Earth 3 Alfred Pennyworth
- Bruce Willis as Victor Fries/Mr. Freeze - A Tragic Villain turned Anti-Hero of the show and Bruce's childhood friend. His goal is to cure his wife Nora Fries and kill Ferris Boyle. Batman's mission is to cure Victor and Nora Fries and also save them from Ferris Boyle.
- Kevin Bacon as Ferris Boyle - The Main Antagonist of the Mr. Freeze arc and Mr. Freeze's arch-enemy. He is a selfish, uncaring, and greedy psychopath who is responsible for Victor's condition. His goal is to kill both Victor, Nora and Batman so no one knows what he did. However, he is finally killed when his heart is frozen solid by Mr. Freeze.
- Jesse Eisenberg as Jervis Tetch/The Mad Hatter - A Minor Antagonist in the season 2 but later Supporting Protagonist in season 3 and a former Wayne Tech employee. This is a younger incarnation of the villain.
- Mark Hamill as The Joker - The Secondary Antagonist of the Christmas special and Batman's arch-enemy.
- Tara Strong as Harleen Quinzel/Harley Quinn - Joker's abused girlfriend. They have an abusive relationship but Batman and the others seems to show sympathy for her while Jason went as far as to try and kill Joker with a crowbar in a fit of rage.
- Tim Roth as Roman Sionis/Blackmask - The Main Antagonist of the Christmas special and Richard Sionis' younger brother. His goal is to avenge his brother's death.
- James Woods as Thomas Wayne Jr./Owlman - The Main Antagonist of both arcs in season 3 and Batman's older half-brother/evil counterpart from Earth-2. His goal is to avenge his brother and parents by destroying the Earth 2 G.C.P.D. (failed).
- Sam Rockwell as Roland Daggett - The Tertiary Antagonist of season 1, The Secondary Antagonist of Season 2, A Minor Antagonist in the Crisis on Two Earths arc, and the Secondary Antagonist of The Court of Owls arc.
- Jeff Bennett as The Jester - The Joker's good counterpart and Owlman's arch-enemy. His goal is to stop Owlman from taking over Earth 2.
- Neil Patrick Harris as The Music Meister - A Singing Villain and The Titular Singing Antagonist of the musical episode The Mayhem Of The Music Meister.
- Helena Bonham Carter as Carolina Greenway/Bat-Venom - The Main Antagonist of the horror-action film. However, she is non-canon which means she is not real.
- Martin Starr as Gil Mason - The Secondary Antagonist of the horror-action film and Carolina's brother. However, he is too non-canon and not real.
- Quinton Flynn as Alberto Falcone - Carmine Falcone's son and Sofia's little brother.
- Jonah Hill as Oswald Copplepot - Owner of the Iceberg Lounge.
- Crispin Glover as Edward Nygma/The Riddler
- Laura Harring as Pamela Isley/Poison Ivy - A former plant doctor and The Riddler's wife.
- Skeet Urich as Billy Maroni - Sal Maroni's deranged son and the Secondary Antagonist of the Circus Of Strange arc of season 4. He is currently incarcerated in Arkham.
- Katrina Bowden as Sofia Falcone - Carmine Falcone's daughter and the Main Antagonist of the Circus Of Strange arc of season 4. Throughout the season, she falls in love with Jason Todd and decides to be his love-interest. However, she ends up blackmailing him in order to help her.
- Dwight Schultz as Lazlo Valentin - A serial killer who turns victims into "Dollotrons". However, he later is revealed to be a bipolar hitman and is actually copyrighting his own son Janosz Valentin, who actually is a serial killer named Professor Pyg.
Special scary episode
The Halloween horror special is called Arkham VR starring Tim Drake as he enters a VR training machine and plays an alternate Batman but the events in the VR story never happened.
The second Halloween horror special is called The Death Of Batman starring Kevin Conroy as the narrator telling or warning us what happens when someone drinks a dangerous chemical. Warning: this special is rated R.
(a flashback shows someone's past. A younger Alberto is home from school but his teenage sister Sofia glares at him)
- Sofia: (angry) "Where the f**k have you been? Do you know what time it is? It's almost midnight, kiddo."
- Alberto: (explaining) "I got beaten up by some jerks from school and fell asleep in some park after waiting for dad."
- Sofia: (annoyed) "You what?"
- Alberto: (tired) "I waited for dad but got beaten up and slept in some park."
(then Sofia gets enraged by this)
- Sofia: (enraged) "Well, I can't even call the cops because you've been waiting on some deadbeat."
- Alberto: (upset) "Look, I didn't mean to."
- Sofia: (annoyed) "You didn't mean to. You forget. This all I've been hearing from you."
(their brother Darren Falcone is hearing this and trying to calm her down)
- Darren: (scared) "Calm down Sofia."
(then Sofia glares at him)
- Sofia: (angry) "You shut up, I'm sick and tired of you keeping him away from me. You hear."
(then Alberto has had enough)
- Alberto: (yelling) "DON'T YOU YELL AT HIM! (an angry Sofia just hits Alberto and causes him to fall on the floor. He just runs into his room, locks the door, and lays on his bed) I hate her. I hate this family. Why can't we just love each other?"
(then he decides to sneak out of his room and runs away from home. In the present day, Carmine hears this story and hugs his son, who hugs back)
- Carmine: (worried) "I'm so sorry that I wasn't there."
- Alberto: (upset) "It's not your fault. All she does is get angry and takes it out on me. I wanted to love her but she's the reason why I don't want to be with my family. She's the reason why I was alone. She's the reason I left home."
(they hug each other. Later when Jason and Pyg are struggling, Sofia makes a choice: Save Jason and keep him as Gotham's hero or to shoot Jason and be saddened by his death. However, an idea goes to her head and instead aims at Pyg)
- Sofia: (smirking) "Love is more better. (they turn to her and she just shoots Pyg, killing him. Jason stares at her in shock) Don't be shocked, Jason. I saved your life. Also, I won't tell anyone your secret unless we become partners. Deal?"
(at first, Jason hesitated but then gives in and shakes her hand)
- Jason: (sighing) "Deal. (however, Sofia yanks him towards her and they share a kiss. Once they let go, Jason takes out his phone) Hello GCPD? This is Red Hood. Pyg is dead. I shot him in self-defense. Were fine."
(Sofia smiles and hugs him, which he returns. Later after Sofia is defeated, Jason smiles at Renee, who smiles back)
- Jason: (smiles) "You're cute when you smile."
- Renee: (also smiles) "You too."
(they then close their eyes and share a kiss)
- Adaptational Heroism: Victor Fries is redeemable along with the Riddler and Poison Ivy. Carmine Falcone and his son also fit in this.
- Adaptational Relationship Overhaul: Unlike the comics, Tim Drake and Barbara Gordon are in love with each other while Riddler is married to Poison Ivy. Harley no longer loves Joker and breaks up with him but falls in love with Deadshot, who loves her back.
- Big Bad: The main villains of the seasons but other villains have this role in other story arcs. Ferris Boyle serves as the main villain of Mr. Freeze's story arc.
- Bound and Gagged: In the first season Joker episode, Barbara Gordon and Renee Montoya are kidnapped by the Joker and tied up with duct tape along with having their mouths taped shut. They are like that whole episode until they are saved. In season 2, Black Canary, Catwoman, and Batgirl become victims of this when they are captured by Matches Malones, Batman's second personality, until Batgirl manages to use her Batarang to cut Canary's gag and it gives her time to use her high pitch boom voice to free them. In season 3, Superwoman is tied up with a construction pipe and gagged with a bandana by her good counterpart Wonder Woman until Flash's counterpart Johnny Quick frees her off-screen. In season 4, Zatanna becomes a victim of this when Sofia captures and gags her because of her magic until the others find her and free her.
- Damsel in Distress: See the Bound and Gagged trope for information.
- Downer Ending: The Circus of Strange Arc ends with Sofia successfully blackmailing Jason Todd into serving her and betrays Lazlo Valentin. Tim and Barbara's relationship is strained when she believes that he is becoming arrogant while Bruce and Jim are losing the support of their friends because of always failing to save others. Also, Billy Maroni is seen about to start plotting with Jerome Valeska to escape from Arkham.
- Earn Your Happy Ending: In "Cold, Cold, Heart", Victor Fries finally managed to cure his wife Nora and got revenge on Ferris Boyle. Sure, he's incarcerated at Arkham but he doesn't mind and is happy. He also is finally cured of his condition and is able to survive without his suit. Even Batman and the others are happy about this ending.
- Fan Service: During a vacation special, Selina, Barbara, and Renee rock the two-piece swimsuits.
- Moral Event Horizon: Joker crosses this by unfairly punishing Harley for his plan failing which was his own fault. Fortunately, Jason punishes him for this.