The story follows Echo (Eliza Dushku), a "doll" or "Active" for the Los Angeles "Dollhouse", one of several fictional facilities run by a company which hires out human beings to wealthy clients. These "engagements" range from romantic interludes to high-risk criminal enterprises. Each Active has their original memories wiped and exists in a child-like blank state until programmed via the insertion of new memories and personalities for each mission. Actives such as Echo are ostensibly volunteers who have surrendered their minds and bodies to the organization for five-year stints, during which their original personalities are saved on hard drives, in exchange for vast amounts of money and a solution to any other problematic circumstances in their lives. Echo is unique however in that she remembers small amounts even after personality "wipes", and gradually develops an increasingly cognizant self-awareness and personality. This concept allows the series to examine the notions of identity and personhood.
Within the house, opinions on such matters are divided. Dollhouse director Adelle DeWitt (Olivia Williams) sees her role as merely giving people what they need; programmer Topher Brink (Fran Kranz) is initially entirely scientific and amoral, apart from brief flashes of moral quandary; while Echo's mentor in the house or "handler", Boyd Langton (Harry Lennix), an ex-cop with an unknown past, is generally concerned with the ethical and theological implications of the Dollhouse's technology, seeing his inside role as an opportunity to limit any collateral damage. Further complicating matters are other dolls Victor (Enver Gjokaj) and Sierra (Dichen Lachman), who despite being continually re-wiped, begin to fall in love.
Meanwhile, FBI agent Paul Ballard (Tahmoh Penikett) learns of Echo's original personality, Caroline Farrell. Agent Ballard becomes obsessed over rumors of the Dollhouse and risks his career trying to prove its existence. As Echo continues to evolve and learns to work beyond the limits of each temporary personality imprint or default "tabula rasa" programming, she runs the risk of being sent to "the Attic", a permanent resting place for "broken" dolls and problematic Dollhouse employees. She is an object of fascination for the escaped doll Alpha (Alan Tudyk) – a genius and serial killer who has been driven mad by being implanted with the memories of dozens of people — who returns at the end of the first season to confront her.
"Epitaph One", the final episode of season one, which was not aired as part of the show's original run on US television, depicts a post-apocalyptic future where the mind-wiping technology of the Dollhouse has spread and brought about the end of civilization. Many of the series' main characters futures are shown. As the second season begins, the show's focus shifts to depict the dangers of abusing the mind-wiping technology. Each character in the L.A. Dollhouse is forced to confront their own moral complicity in an increasingly downward spiral from moral grey areas to the realization that what the Dollhouse is doing is ultimately immoral and wrong. The Dollhouse's corporate sponsor is a medical research entity known as the Rossum Corporation, whose ultimate goal is revealed to be gaining control over national governments and later even innocent people with no association with the Dollhouse. Attempting to stop the further spread of the mind-wiping technology, the L.A. Dollhouse vows to take down Rossum and its mysterious founder whom only Echo's original personality, Caroline, has met.
The final episode of the series is set in the year 2020, leading on from "Epitaph One". Despite its best efforts, the L.A. Dollhouse has been unsuccessful in stopping the mind-wiping technology from spreading out of control. Rossum executives use multiple bodies to live in decadence while the peoples of the world are enslaved. A now deranged Topher, architect of much of the technology, devises a way of ending its spread, but at great sacrifice to himself and others. The series concludes with the world's personalities restored, while the Earth still lies in ruins, and those with Active architecture locked inside the Dollhouse for at least two years.