Objective To study the flavonoids from Daphne pedunculata H. F. Zhou ex C. Y. Chang. Methods The compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20 gel column chromatography and semi-HPLC, and their structures were elucidated by NMR and mass spectrometry. Results 21 flavonoids were isolated from the 95% ethanol extract of Daphne pedunculata, including 12 biflavones. They were identified as isochamaejasmin (1), neocohamaejasmin A (2), neocohamaejasmin B (3), wikstrol B (4), daphnodorin D2 (5), wikstrol A (6), genikwanol A (7), daphnodorin B (8), wikstaiwanone B (9), wikstaiwanone A (10), daphnodorin G (11), apigenin (12), luteolin (13), genkwanin (14), astragalin (15), apigenin-7-O-glucoside (16), kaempferol (17), luteolin-7-O-glucoside (18), acacetin (19), daphnodorin F (20), 5-methoxy-apigenin-7-O-β-D-glucoside (21). Conclusion Among the 21 flavonoid compounds isolated from the 95% ethanol extract of Daphne pedunculata, except compounds 12~14, the others were isolated from Daphne pedunculata for the first time. The research results enrich the chemical diversity of flavonoids in Daphne pedunculata, and provide an important foundation and scientific basis for its in-depth research and utilization.