Abstract: To investigate the inhibitory activity and mechanism of genkwanin on α-glucosidase, the inhibitory activity was determined by ultraviolet and fluorescence spectroscopy, including the inhibitory activity, inhibitory type of genkwanin on α-glucosidase and the fluorescence quenching properties. The results showed that the genkwanin had a significant inhibitory effect on α-glucosidase in a competitive manner. Genkwanin interacted with α-glucosidase was mainly driven by hydrogen bond and Van der Waals force with the binding sites of 2.8 and 2.2 at 291 and 310 K, respectively. The formation of genkwanin and α-glucosidase complexes resulted in fluorescence quenching of α-glucosidase and thereby inhibited the activity of α-glucosidase.