The title of the series refers to the burn notices issued by intelligence agencies to discredit or announce the dismissal of agents or sources who are considered to have become unreliable. When spies are burned, their connection to an espionage organization is terminated, leaving them without access to cash or influence. According to the narration during the opening credits, the burned spy has no prior work history, no money, no support network – in essence, no identity. The television series uses second-person narrative and frequent voice-overs providing exposition from the viewpoint of covert operations agent Michael Westen, played by Donovan. The voice-over commentary is in the form of tips for fledgling agents as if for a training or orientation film.

After being "burned" in the middle of an operation in southern Nigeria and subsequently beaten and kidnapped, Westen finds himself in his hometown of Miami, Florida. He is tended to by his ex-girlfriend, Fiona Glenanne (Gabrielle Anwar), but he has been abandoned by all his normal intelligence contacts and is under continuous surveillance with his assets frozen. Extraordinary efforts to reach his U.S. government handler eventually yield only a grudging admission that someone powerful wants him "on ice" in Miami. If he leaves there, he will be hunted down and taken into custody. If he stays, he can remain relatively free. Consumed by the desire to find out who burned him and why Westen is reluctantly drawn into working as an unlicensed private investigator and problem solver for ordinary citizens to fund his investigation into his situation as a blacklisted agent.

Westen invites his old friend Sam Axe (Campbell) to assist him, while Fiona invites herself to join them. With the occasional assistance and sometimes hindrance of his mother, Madeline (Sharon Gless), Westen battles an array of criminals such as mobsters, gang members, con artists, murderers, rapists, kidnappers, foreign wet-work operatives, drug traffickers, sex traffickers, arms traffickers, and war criminals. At the same time, Michael must follow the trail that leads him to the people responsible for his being burned and later finds out why.

The series juggles these two narratives: the overall series dealing with why Michael was burned, and individual episodes focusing on the cases he works for clients.