In this work, the aqueous two-phase extraction parameters and in vitro antioxidant activity of total flavonoids from Ginkgo biloba were investigated. The partition behavior of the flavonoids in an aqueous two-phase system (ATPS) was analyzed, the extraction parameters were optimized using response surface methodology, and the antioxidant activity of the flavonoids was determined by free-radical scavenging tests (1,1-diphenyl-2-picrylhydrazyl (DPPH) assay) and the ferric reducing/antioxidant power (FRAP) assay. The results showed that the concentration of ammonium sulfate was negatively correlated with the phase-volume ratio, whereas the concentration of polyethylene glycol was positively correlated. The maximum yield of flavonoids (4.11 mg g−1) was obtained under the following optimal extraction conditions: Concentration of polyethylene glycol (PEG)1500 30% and (NH4)2SO4 22% (mass fraction), and liquid/solid ratio 40:1 (mL g−1). The antioxidant activity tests showed that the flavonoids from the G. biloba leaf exhibited free-radical scavenging activity, with an IC50 of 2.66 mg L−1, which was superior to that of vitamin C. The free-radical scavenging ability of the flavonoids was proportional to the flavonoid concentration. The total reducing power of the Ginkgo flavonoids was slightly lower than that of vitamin C. In this study, the distribution of flavonoids in an ATPS was analyzed and a mathematical model for the ATPS extraction of Ginkgo flavonoids was established, which provides a reference for further development and utilization of G. biloba.