A method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (ESI) was established for the separation and characterization of flavonoids in Sophora flavescens Ait. Based on the chromatographic separation of most flavonoids present in S. flavescens Ait., a total of 24 flavonoids were identified. Fourteen compounds were unambiguously identified comparing experimental data for retention time (t_R), UV and MS spectra with those of the authentic compounds: 3′,7-dihydroxy-4′-methoxy-isoflavone (13), trifolirhizin (14), kurarinol (18), formononetin (19), 7,4′-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (22), maackiain (21), isoxanthohumol (23), kuraridine (26), kuraridinol (27), sophoraflavanone G (30), xanthohumol (31), isokurarinone (33), kurarinone (35) and kushenol D (38), and additional 10 compounds were tentatively identified as kushenol O (10), trifolirhizin-6′′-malonate (15), sophoraisoflavanone A (20), norkurarinol/kosamol Q (24), kushenol I/N (25), kushenol C (28), 2′-methoxykurarinone (29), kosamol R (32), kushecarpin A (34) and kushenol A (37) by comparing experimental data for UV and MS spectra with those of literature. Furthermore, fragmentation pathways in positive ions mode of 24 flavonoid compounds of types of flavanone, flavanonol, flavonol, chalcone, isoflavone, isoflavanone and ptercocarpane were summarized. Some common features, such as CH₃·, H₂O, CO, CO₂, C₃O₂ and C₂H₂O losses, together with Retro-Diels–Alder fragmentations were observed in the prenylated flavonoids in S. flavescens Ait. The loss of the lanandulyl chain was their characteristic fragmentation, which might help deducing the structure of unknown flavonoid compounds. The present study provided an approach to rapidly characterize bioactive constituents in S. flavescens Ait.