The series is a continuation of its predecessor, taking up soon after Justice League ended. It features a greatly-expanded League, in which the characters from the original series—now referred to as "founding members"—are joined by a large number of other superheroes from the DC Universe; in the first episode, well over 50 characters appear. A number of these were heroes who had made guest appearances in the first two seasons of Justice League, Batman: The Animated Series, and Superman: The Animated Series, but many heroes and other characters made their first animated appearances in this series. The general format of each episode is to have a small ad hoc team assemble to deal with a particular situation, with a focus on both action and character interaction. The major theme is a battle of Government versus Super Heroes according to the Superhuman Registration Act.

Most episodes tell a self-contained story, but the series also features extended story arcs, the first involving the building conflict between the League and a secret government agency known as Project Cadmus. This plot line builds upon events that occurred during the second season of Justice League (which in turn built upon events in Batman: The Animated Series, Superman: The Animated Series, Batman Beyond, Static Shock and The Zeta Project), and has affected the plotlines of most of its episodes. It was resolved in a four-part story at the end of the second season of Justice League Unlimited. The third season story arc focuses on the new Secret Society (which is based on the Legion of Doom) as the main villains, a loose-knit organization formed to combat the increased superhero coordination of the first season.