Combined with molecular docking model, a fluorescence method was applied to investigate the interaction between quercetin and β-glucosidase and the acting mechanism. The interaction between β-glucosidase and quercetin, as well as the enzyme inhibitor 4-nitrophenyl-β-D-thioglucoside, was studied by the AutoDock4.2 molecular docking model, respectively. The binding reaction was simultaneously studied using fluorescence quenching method. The results showed that these interactions result in the endogenous fluorescence quenching of β-glucosidase, which belongs to a static quenching mechanism. The calculated binding constants were 4.36×104, 4.04×104 and 3.18×104 L·mol-1 at 17, 27 and 37 ℃, respectively. The results revealed that quercetin tended to bind with β-glucosidase mainly by hydrogen bond and hydrophobic interaction, as well as electrostatic forces. Both fluorescence spectroscopy and molecular docking are complementary to each other for the investigation of the interaction between β-glucosidase and quercetin from the experimental and theoretical view.