Hypericum attenuatum Choisy is a traditional Chinese herbal plant with multiple therapeutic effects. In this study, bioactivity‐guided fractionation of Hypericum attenuatum Choisy extracts afforded three major flavonoids (including astragalin, guaijaverin and quercetin), which possessed α‐Glucosidase inhibitory activity with IC50 values of 33.90±0.68 μM, 17.23±0.75 μM and 31.90±0.34 μM, respectively. Circular dichroism analysis revealed that all the three compounds could interact with α‐glucosidase by inducing conformational changes of the enzyme. Molecular docking results indicated that they could bind to the active site in α‐glucosidase, and the binding force was driven mainly by hydrogen bond. Additionally, isobolographic analysis of the interactions between two compounds showed that all the combinations presented a synergistic α‐glucosidase inhibitory effect at lower concentrations, and the combination between quercetin and guaijaverin or astragalin exhibited the best synergistic effect. This research might provide a theoretical basis for the application of Hypericum attenuatum Choisy in treating hyperglycemia.