The story follows Echo, a "doll" or "Active" for the Dollhouse, an organization which hires out reprogrammable human beings to wealthy clients who use them for a range of purposes, such as sexual encounters and high-risk illegal activities. 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 surrender their bodies to the organisation for five year stints, during which their original personalities will be saved, in exchange for vast amounts of money and a solution to any other extenuating circumstances in their lives. Echo, however, is unique in remembering 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 graduate 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 her current personality imprint or default programming, she runs the risk of going to "the Attic", a place for broken dolls. She is an object of fascination for the escaped doll Alpha (a genius and serial killer who sees Echo as a potential mate) and FBI Agent Paul Ballard, whose obsession with the urban legend that is the Dollhouse costs him his career, before he comes to work for the organisation as Echo's bodyguard or "handler". Ballard sees the Dollhouse's activities as immoral and illegal, but becomes increasingly complicit in the business which he equates with murder and sex traffic. Within the house, opinions are divided; director Adelle DeWitt sees her role as honourable, programmer Topher Brink's view is entirely scientific and amoral, and handler-turned-head of security Boyd Langton, like Ballard, is more concerned with the ethical and theological implications of the Dollhouse's technology.