Methods of purifying a virus from a virus-infected cell lysate using three-phase partitioning (TPP) are disclosed. The methods comprise a first round of TPP, including mixing a cell lysate comprising a virus with ammonium sulfate and t-butanol, and separating the mixture, thereby forming a first aqueous phase, a first organic phase, and a first interphase. The first aqueous phase can comprise the virus, which can be subjected to a second round of TPP, resulting in a second aqueous phase, a second organic phase, and a second interphase. The second interphase can comprise highly purified virus. The methods can comprise subjecting a first aqueous phase to further purification by column chromatography or density gradient centrifugation. Purification of AAV, including AAV2, AAV5 and AAV6, from lysates of infected insect cell cultures is demonstrated. TPP-purified AAV particles infect at least as well as those prepared by standard methods.