To evaluate the development potential of Tartary buckwheat flavonoids (TBF) as natural inhibitors of xanthine oxidase (XO) activity, this study investigated the inhibitory effect and mechanism of TBF on XO activity through enzyme inhibition assays, molecular docking, and molecular dynamics simulation. The results showed that TBF exhibited good inhibitory effects on XO activity, with a half-maximal inhibitory concentration (IC50) of 0.83 mg•mL-1. At a TBF concentration of 1.4 mg•mL-1, the inhibition rate reached 60.43%, and inhibition followed a reversible mixed mode. The main TBF components were rutin and quercetin. The active binding sites of rutin with XO included PHE1142, VAL1011, PHE1013, LEU1014, and PHE649, primarily via hydrogen bonding and π-π interactions, with a binding free energy of −26.42 kcal•mol-1. The active binding sites of quercetin with XO included LEU648, VAL1011, PHE649, SER876, and ASN768, primarily via hydrophobic interactions, π-π bonds, and hydrogen bonding, with a binding free energy of -27.70 kcal•mol-1. Both rutin and quercetin formed stable complexes with XO, thereby effectively inhibiting XO-catalyzed oxidation of purine to uric acid. This study provides evidence for the enzymatic and molecular mechanisms by which TBF inhibits XO activity, indicating its potential for developing functional foods that regulate uric acid metabolism and help prevent hyperuricemia and gout.