Flavonoids, also called vitamin P, are widely distributed in plants fulfilling many functions. Yeast α- glucosidase (YAGH; EC 22.214.171.124), as extensively used target protein for screening bioactive compounds from medicine plants, was selected to explore the possible mechanisms of multiple biological function of flavonoids. The results in this study indicated that flavonoids, as mixed-type inhibitors, quenched the intrinsic fluorescence of YAGH by a mixed fluorescence quenching mechanism. The interaction information between flavonoids and YAGH was analyzed using a flexible docking method (AutoDock) and showed that 3, 4 dihydroxyl groups of B ring and 3-OH of C ring played a more important role in the inhibition activity than other hydroxyl groups, because the 3, 4 dihydroxyl groups of B ring directly interacted with the active-site residues of YAGH to inhibit enzyme activity and 3-OH of C ring seemed to be necessary to maintain the proper binding orientation of flavonoid molecules, thereby making the hydroxyl groups of B ring interact with active-site residues tightly in the hydrophobic pocket of YAGH. The results supply a basis for understanding the mechanisms of multiple biological functions of flavonoids.