The inhibitory conditions and mechanisms by which cyanidin-3-O-glicoside (C3G) acts on the catalytic activity of heme were examined in this study. Using spectroscopic and chromatographic analyses, the optimal pH values for heme-catalyzed production of hydroxyl radicals (•OH) from H2O2 to oxidize three substrates-2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 3,3′,5,5′-tetramethylbenzidine (TMB), and O-phenylenediamine (OPDA)-were 4.5, 5.5, and 4.0, respectively, with optimal temperatures of 50, 40, and 50 ℃ , respectively. Kinetic analysis indicated that the heme had the highest affinity for ABTS (Km=0.37 mmol•L−1) and the fastest catalytic rate for TMB (Vmax=1.415×10−2 mmol•L−1•min−1). At 50 ℃ and pH 4~5, the IC50 values of C3G for inhibiting the oxidation of the three substrates were 0.235, 1.537, and 0.305 mmol•L−1, respectively. C3G (50~300 μmol•L−1) exhibited a molar concentration-dependent inhibitory effect on heme (20 mmol•L−1) catalytic activity. Kinetic analysis further revealed that this inhibition was reversible and competitive. As the C3G concentration increased, the Km value gradually increased while Vmax remained essentially unchanged, with an inhibition constant (Ki) of 2.549 mmol•L−1. Further investigation into the inhibition mechanism showed that C3G could scavenge •OH generated by heme-catalyzed H2O2. At equivalent concentrations, C3G demonstrated stronger •OH scavenging activity (IC50=0.622 mmol•L−1) compared with ascorbic acid (IC50=1.076 mmol•L−1). Ultraviolet spectroscopy and liquid chromatography studies indicated that C3G binds to heme, resulting in the formation of new compounds. Collectively, these results demonstrate that C3G exerts a strong inhibitory effect on heme catalytic activity under specific conditions, thus providing a theoretical foundation for the utilization of C3G-rich food resources and the development of antioxidant functional foods aimed at preventing or mitigating heme-induced oxidative damage.