Interaction of flavonoids and enzyme may affect characteristics and physiological activities of both components. In this study, the effects of the interaction between four flavonoids (quercetin, luteolin, kaempferol and apigenin) and trypsin were examined. At the concentration of 2.7 mm, inhibition of trypsin (1.6 U mL−1) by quercetin, luteolin, kaempferol and apigenin was 46.4%, 32.6%, 26.8% and 17.7%, respectively. In the presence of trypsin, DPPH, ABTS and hydroxyl radical scavenging activities of flavonoids were obviously inhibited. Addition of flavonoids led to fluorescence quenching of trypsin. The decreasing order of binding force between flavonoids and trypsin was quercetin, luteolin, kaempferol and apigenin. It is concluded that the interaction between flavonoids and trypsin depends on the number and position of hydroxyl group of flavonoids.