While investigating the events that led to their mentor's demise, the heroes become embroiled in an ancient conflict between two secret organizations fighting for control of Gotham: the Court of Owls and the League of Shadows.

Gameplay is presented from the third-person perspective with a primary focus on using each playable character's traversal and combat abilities to defeat enemies and explore the environment.

[6] While the narrative campaign can be played solo, the game also features a two-player cooperative multiplayer mode, wherein the second player can drop in and out at any time without affecting the other one.

[15] During the game, the Knights come into conflict with several supervillains, primarily the Court of Owls, a secret society dating back to colonial times that seeks to take over Gotham with their legion of assassins called Talons, and the League of Shadows, led by Talia al Ghul (Emily O'Brien) after the death of her father Ra's al Ghul (Navid Negahban).

Freeze (Donald Chang),[16][17] Harley Quinn (Kari Wahlgren),[18] Clayface (Brian Keane),[19] the Man-Bat Commandos, and Penguin (Elias Toufexis).

[23] Supporting characters include Bruce's former butler Alfred Pennyworth (Gildart Jackson),[24] who provides technical assistance and moral support to the Knights; police captain Renee Montoya (Krizia Bajos),[25] one of the few honest cops left in Gotham;[26] Bruce's uncle and wealthy industrialist Jacob Kane (Tommie Earl Jenkins) and his wife Commissioner Catherine Kane (Liz Burnette); former WayneTech employee-turned-CEO of Foxteca Lucius Fox (Peter Jay Fernandez); Gotham City News anchorwoman Noor Rashid (Zehra Fazal); journalist Arturo Rodriguez (Mateo d'Amato), Gotham City Mayor Brandon Tsai (David Shih); and Jada Thompkins (Keisha Castle-Hughes), daughter of Leslie Thompkins who took up her mother's profession as a doctor after she retired.

With the assistance of Alfred Pennyworth following the funeral attended by Bruce's uncle Jacob Kane, the Knights get the Belfry operational and pursue one of Batman's leads, a scientist named Kirk Langstrom.

When they infiltrate the GCPD morgue to recover Langstrom's DNA encryption key, they encounter Talia al Ghul who incinerates Ra's' body and warns the Knights the League of Shadows will make their move on Gotham soon.

The Knights recover her intelligence and discover evidence of a centuries-long conspiracy where multiple violent criminals were released early from Blackgate after witnesses to their crimes were mysteriously murdered, with Langstrom being the latest victim.

The Penguin tells them of the existence of the Court of Owls, a secret society that supposedly rules Gotham from the shadows, and directs the Knights to investigate the ultra-elite Powers Club.

While learning that Penguin's mother Constance Cobblepot is a member, the Knights discover the identity of their leader (who holds the title of Voice of the Court) and are shocked to find out it is Jacob Kane.

Having gathered enough evidence to implicate Kane, the Knights enlist the aid of Renee Montoya, a trustworthy GCPD detective, revealing their true identities to her as a sign of trust.

Talia reveals she manipulated the Knights into dismantling the Court for her and now plans to lead the League to purge Gotham of all corruption.

The Knights follow Talia's trail to the ruins of Arkham Asylum, where they find that the League has appropriated Langstrom's research and Lazarus Pit water to create mutant Man-Bats.

After dealing with the Man-Bats, the Knights follow the League into a network of tunnels underneath the Gotham Cemetery, where they find a Lazarus Pit under the ruins of the Batcave.

To reassure the people of Gotham while planning to expose Kane, the Knights make a public address declaring that the city is under their protection.