The philanthropist and playboy, Bruce Wayne, and presidential candidate, Lex Luthor, are both nominated for the Man-of-the-Year award, which goes to Bruce. Five villains-The Joker, Harley Quinn, The Penguin, The Riddler, and Two-Face- along with henchmen raid the event and begin stealing from the guests. Bruce exits stage-right and changes into his Batman costume . Joker takes the trophy and begins his "acceptance speech", but is interrupted by Batman and Robin crashing through the stage in the Bat-boat. Four of the villains exit through the trapdoor, leaving Harley Quinn to cover their flight. She is defeated and the Dynamic Duo begin hunting the other villains in the theatre. However, The Joker escapes.
Batman and Robin chase him in the Batwing and Robin's helicopter, and defeat him outside of the Gotham Funland. After the police haul him off, SuperMan arrives to greet the Dynamic Duo, apologize to "Bruce" for missing the ceremony, and to scold Batman for making a mess at the theatre.
During the raid, Lex earned an admiration for The Joker, and the two form an alliance later when Lex breaks Joker out of Arkham. With The Decounstructor, they can destroy shiny, black objects (such as the walls of Penguin's cell), and Lex hints at using it on Batman's vehicles. While in Luthor's helicopter, Joker begins aiming the Decounstructor at different cells and frees Catwoman, The Riddler, Two-Face, Harley Quinn, Poison Ivy, and Bane.
Meanwhile, in the Batcave, Bruce Wayne is contacted by Martian Manhunter from the Justice League Watchtower and informs him of an emergency. Alfred shows Bruce and Dick Grayson where it is on a map, and they change into Batman and Robin and leave for Arkham Asylum where they find the villains Joker freed attempting a prison break. They then attempt to stop them, starting with Catwoman. The villains are stopped, and the police arrive. Commissioner Gordon informs the Dynamic Duo that all of the villains have been accounted for, except Joker. Batman notices an electrified brick outside of Joker's cell. He and Robin go to investigate. They run-in to Killer Croc and Mr. Freeze, who create obstacles, and stop The Scarecrow from escaping. They bring a black piece back to the Batmobile and discover it was destroyed with LexCorp technology and conclude that Luthor broke Joker out. Batman, Robin, and Gordon are alerted by a Police Officer that there has been a break-in at Ace Chemicals, and they leave. When Gordon and the Officer look back, they realize that the villains have escaped and that other officers are tied up in their places. Two-Face and Riddler wave at them as they escape in a police van.
At Ace Chemicals, Batman and Robin find that The Joker had already been there and stolen chemicals. They try to escape when a fire begins and just before they fall into the inferno, they are rescued by Superman. With help from Superman, they are able to escape. Batman tries to find out what Joker is making, and realizes it is Kryptonite. He uses the Batcomputer to find nearby locations of Kryptonite, and heads downtown. They follow a large vehicle, the Juggernaut. On the inside, Lex and Joker are creating Kryptonite. Batman and Robin begin firing at it. When the defenses are weakened enough, the Dynamic Duo infiltrate it. Batman takes the Kryptonite, and Lex is subdued. Joker however uses his joybuzzer on Batman and Robin to the point where they fall out of the Juggernaut, into their vehicles. Joker aims the Deconstructor at the Batmobile, which falls apart, and the Juggernaut gets away.
Back in the Batcave, Batman analyzes the Kryptonite and finds that it is only similar to true Kryptonite and that is harmless to Kryptonians. However, if there wasn't actual Kryptonite in the Juggernaut, the Batmobile's computer shouldn't have shown it. He and Robin decide that it was just a trick of Joker's, and put it away with the other Kryptonite they store in-case Superman goes bad.
Meanwhile, in the Juggernaut, Lex and Joker who are tracking the location of the false Kryptonite lose their signal, but realize that it is directly ahead. They use the Deconstructor to open a cavern and enter the Batcave. A fight ensues, and the Bat-bike, Batwing, and Batboat are destroyed. Batman and Robin flee through the elevator, and hang on to the shaft to escape when it falls. Joker and Lex find the Kryptonite vault and leave with it after Joker's pies explode and a fire begins. At the top of the elevator shaft, Superman arrives to rescue Batman and Robin.
Joker and Lex's alliance is threatening enough that it grabs the attention of Justice League members such as Superman, Wonder Woman, and Green Lantern who each team-up with the Dynamic Duo at some point.
Near the end of the game, Lex Luthor is persecuted, but Joker still has access to a Kryptonite powered mech. He chases Batman and Robin through Gotham City, before he attacks WayneTech. At the top of the tower, Superman and Wonder Woman are preparing to defend it. Batman , Robin , Green Lantern and Cyborg enter through the bottom of the building together.
Though not a part of the main story, there are side-missions around Gotham City to rescue endangered civilians and to locate and defeat hidden villains, such as Bane on the beach or The Penguin at the zoo's Arctic World exhibit.
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