The inhibitory mechanism of α-amylase and α-glucosidase by cyanidin-3-O-glucoside was investigated by ultrafiltration, high performance liquid chromatography (HPLC), enzyme kinetics, and molecular docking. The results indicated that cyanidin-3-O-glucoside inhibited α-amylase and α-glucosidase in a reversible and non-competitive manner. Besides, the fluorescence quenching analysis indicated that cyanidin-3-O-glucoside combined with the two enzymes by hydrogen bonds to form a complex. Molecular docking analysis showed that cyanidin-3-O-glucoside interacted with the key amino acid residues of α-amylase and α-glucosidase through hydrogen bonds and hydrophobic forces, and the binding energies were-7.8 and-9.8 kcal/mol, respectively. Our research suggests that cyanidin-3-O-glucoside has the potential to be used as an inhibitor of α-amylase and α-glucosidase in the development of functional foods.