Flavonoids can regulate blood sugar levels by inhibiting the activity of α-amylase. However, there were very limited research on the inhibitory mechanism and structure-activity relationship of flavonoids on α-amylase. Especially, the effects of hydroxylation on the B ring and glycosylation on the C ring related to the inhibitory ability of flavonoids on α-amylase were still unclear. Therefore, in order to explore the inhibitory mechanism and structure-activity relationship of flavonoids on α-amylase, the types of inhibition, fluorescence quenching properties, conformational changes, binding force and binding sites of three flavonoids (rutin, quercetin and kaempferol) were investigated in this study. The results showed that kaempferol had the strongest ability to inhibit α-amylase and rutin had the weakest ability to inhibit α-amylase, respectively. The inhibitory effect of flavonoids was attenuated by glycosylation at C3 on the C ring and hydroxylation at C3' on the B ring. The quenching affinity was enhanced by a glycosyl group at C3 on the C ring and weakened by a hydroxyl group at C3' on the B ring. Hydrogen bonds and hydrophobic interactions caused the spontaneous binding between the flavonoids and α-amylase, which changed structure and hydrophobic microenvironment of α-amylase, and inhibited α-amylase activity effectively. The purpose of this study was to provide theoretical support for the development and application of hypoglycemic foods rich in flavonoids.