The show revolves around a corporation running numerous underground establishments (known as "Dollhouses") across the globe which program individuals referred to as Actives (or Dolls) with temporary personalities and skills. Wealthy clients hire Actives from Dollhouses at great expense for various purposes. The series primarily follows the Active known as Echo, played by Eliza Dushku (who also acts as producer), on her journey towards self-awareness.

Dollhouse has received mixed reviews; Robert Bianco of USA Today describing it as a show "that Joss Whedon's most devoted fans will debate and embrace, and a mass audience just won't get."[6]

The story follows Echo, a "doll" or "Active" for the Los Angeles "Dollhouse", one of several facilities that is run by an organization that hires out human beings to wealthy clients who use them for a range of fantasy purposes, called engagements. Engagements range from romantic interludes to high-risk criminal enterprises. The dolls' memories have been wiped using sophisticated technology, and new memories and personalities are programmed into them for each of their jobs. All of the dolls at the LA Dollhouse are referred to by codenames based on the NATO phonetic alphabet. Echo, like her fellow dolls Victor and Sierra, exists in a child-like blank state, until a programmer uploads her with the skills and memories to make her a completely new and unique person. 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. In reality, however, it appears that heavy coercion or brute force has been used in a number of cases. Echo is unique in that she remembers small amounts even after personality "wipes", and gradually develops an increasingly cognizant self-awareness and personality. This emerging personality is even distinct in some ways from that of her original identity, college student Caroline Farrell. This concept allows the series to examine the notions of identity and personhood.

As Echo continues to evolve and learn 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, a genius and serial killer who has been driven mad by being implanted with the memories of dozens of people and who sees Echo as a potential mate, and FBI Agent Paul Ballard, whose obsession with the unsubstantiated rumours of the Dollhouse costs him his career. An ethical dilemma and persecution by his employer drives Ballard to leave the FBI and work for the organization as Echo's bodyguard or "handler". Ballard sees the Dollhouse's activities as immoral and illegal, but becomes increasingly complicit in the business that he equates with murder and sex trafficking. Within the house, opinions are divided. Director Adelle DeWitt sees her role as aiding an honorable research arm of the Dollhouse's corporate sponsor and giving people what they need; programmer Topher Brink's view is entirely scientific and amoral, apart from brief flashes of moral quandary; and handler-turned-head of security Boyd Langton, an ex-cop with a mostly-unknown past, is generally concerned with the ethical and theological implications of the Dollhouse's technology but seems to view his inside role as an opportunity to limit the collateral damage.