The study investigates the inhibitory activity of six groups of flavonoids against yeast and rat small intestinal α-glucosidases, and porcine pancreatic α-amylase. The results show that anthocyanidin, isoflavone, and flavonol groups are potent inhibitors of yeast α-glucosidase, with IC50 values less than 15 μM. The presence of an unsaturated C ring, 3-OH, 4-CO, linkage of the B ring at the 3 position, and hydroxyl substitution on the B ring enhance inhibitory activity. Rat small intestinal α-glucosidase is weakly inhibited by many flavonoids, with slight inhibition by anthocyanidin and isoflavone groups. 3-OH and hydroxyl substitution on the B ring increase inhibitory activity. In porcine pancreatic α-amylase, luteolin, myricetin, and quercetin are potent inhibitors with IC50 values less than 500 μM. The 2,3-double bond, 5-OH, linkage of the B ring at the 3 position, and hydroxyl substitution on the B ring enhance inhibitory activity, while 3-OH reduces it. The structure-activity relationship is discussed, highlighting the importance of specific chemical structures in the inhibitory activity of flavonoids.The study investigates the inhibitory activity of six groups of flavonoids against yeast and rat small intestinal α-glucosidases, and porcine pancreatic α-amylase. The results show that anthocyanidin, isoflavone, and flavonol groups are potent inhibitors of yeast α-glucosidase, with IC50 values less than 15 μM. The presence of an unsaturated C ring, 3-OH, 4-CO, linkage of the B ring at the 3 position, and hydroxyl substitution on the B ring enhance inhibitory activity. Rat small intestinal α-glucosidase is weakly inhibited by many flavonoids, with slight inhibition by anthocyanidin and isoflavone groups. 3-OH and hydroxyl substitution on the B ring increase inhibitory activity. In porcine pancreatic α-amylase, luteolin, myricetin, and quercetin are potent inhibitors with IC50 values less than 500 μM. The 2,3-double bond, 5-OH, linkage of the B ring at the 3 position, and hydroxyl substitution on the B ring enhance inhibitory activity, while 3-OH reduces it. The structure-activity relationship is discussed, highlighting the importance of specific chemical structures in the inhibitory activity of flavonoids.