Flavonoids in plants used for the treatment of various cardiovascular, cancer diseases have been reported to possess potential protective effects against oxidative injury. Ginkgo biloba leaves, known for their antioxidant activity, were chosen for this study. In this paper, 12 flavonoids in G. biloba leaves were identified by HPLC-diode array detection (DAD)-electrospray ionization MS. HPLC-DAD coupled with chemiluminescence detection was used to determine free radical scavenging activity of flavonoids. It was found that the flavonol glycosides could markedly inhibit the luminescent signal, which indicated that they are mainly responsible for the antioxidant activities of G. biloba leaves. Total antioxidant activity of these flavonoids was used to evaluate the differences of G. biloba leaves collected in 13 habitats. The combination of chemical and activity analysis can provide a valid method to quantify the bioactive components in G. biloba leaves, and this may be a more rational approach to the quality assessment of G. biloba leaves.