Captain Malcolm 'Mal' Reynolds is a former galactic war veteran who is the captain of the transport ship "Serenity". Mal and his crew, ensign Zoe Alleyne Washburne; Zoe's husband, pilot Hoban 'Wash' Washburne; muscular mercenary Jayne Cobb; young mechanic Kaylee Frye; former Alliance medical officer Simon Tam; his disturbed teenage sister River (both on the run from the interplanetary government "The Alliance"); the beautiful courtesan Inara Serra; and preacher Shepherd Book do any jobs, legal or illegal, they can find as the Serenity crew travels across the outskirts of outer space.

The show takes its name from the "Firefly-class" spaceship Serenity that the central characters call home. It resembles a firefly in general arrangement and the tail section, analogous to a bioluminescent insectoid abdomen, lights up during acceleration. The ship was named after the Battle of Serenity Valley, where Sergeant Malcolm Reynolds and Corporal Zoe Alleyne were among the survivors on the losing side. It is revealed in "Bushwhacked" that the Battle of Serenity Valley is widely considered to have sealed the Independents' fate.

The Alliance is shown to govern the star system through an organization of "core" planets, following its success in forcibly unifying all the colonies under one government. DVD commentary suggests that the Alliance is composed of two primary "core" systems, one predominantly Western, the other pan-Asian, justifying the mixed linguistic and visual themes of the series. The central planets are firmly under Alliance control, but the outlying planets and moons resemble the American Old West, under little governmental authority. Settlers and refugees on the outlying worlds have relative freedom from the central government but lack the amenities of the high-tech civilization that exists on the inner worlds. The outlying areas of space ("the black") are inhabited by the Reavers, a cannibalistic group of nomadic humans.

The captain of Serenity is Malcolm "Mal" Reynolds (Nathan Fillion). The episode "Serenity" establishes that the captain and his first mate Zoe Washburne, née Alleyne (Gina Torres) are veteran "Browncoats" of the Unification War, a failed attempt by the outlying worlds to resist the Alliance. A later episode, "Out of Gas", reveals that Mal bought the spaceship Serenity to live beyond Alliance control. Much of the crew's work consists of cargo runs or smuggling. The main story is that of River Tam (Summer Glau) and her brother Simon (Sean Maher). River is a child prodigy whose brain was subjected to Alliance scientists at a secret government institution; she displays symptoms of schizophrenia and often hears voices. It is later revealed that she is a "reader", one who possesses telepathic abilities. Simon gave up a career as an eminent trauma surgeon in an Alliance hospital to rescue her, and they are fugitives. In the original pilot, "Serenity", Simon joins the crew as a paying passenger with River smuggled on board as cargo. As Whedon states in an episode of a DVD commentary, every show he does is about creating a family. By the last episode, "Objects in Space", the fractured character of River has finally become whole, partly because the others decided to accept her into their "family" on the ship.