Abstract: Ginkgo biloba is one of the best explored herbal drugs which could be widely used in the treatment of cognitive decline and Alzheimer’s diseases. Ginkgo biflavones is a kind of Ginkgo biloba flavone and only existed in the its leaves. As the complex structure of the Ginkgo biflavones, the further analysis and summary of this kinds of constituents were few reported. In this experiment, mass spectrometric fragmentation pathways of three Ginkgo biflavones (Bilobetin, Sciadopitysin, Isoginkgetin) were studied. Three standard Ginkgo biloba flavonoids were prepared into a mixture of 20 mg/L for injection. High performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS) was used to collect data from samples with different collision energy at negative ion mode. With the optimizing study of the collision energy, 50 eV was selected for the further study as it could provide more abundant fragmentation information. Based on the high resolution mass spectrum data, the fragmentation pathways of the three Ginkgo biloba flavonoids were summarized. As the structural characteristics of bisflavones, the neutral loss of CH₃OH and CO₂ were easily observed. The major fragmentation pathway was Retro-Diels-Alder (RDA) reaction which could occurs on Ⅰ—C and Ⅱ—C rings in three types, and the substituents could be further lost to obtain the corresponding characteristic fragment ions. The analysis of fragmentation pathway can provide a basis for the structural identification of Ginkgo biloba flavonoids. Moreover, it provides a rapid method to characterize the flavonoids in traditional Chinese medicine (TCM) by HPLC-Q-TOF MS which also can be used in searching for new constituents and their metabolites in vivo.