Xanthine oxidase (XO) is an important target for the treatment of hyperuricemia (HUA), which mediates the production of uric acid (UA). The mechanism of action between flavonoids and XO in cell biology and structural bioinformatics has not been fully explored. In this study, 160 flavonoids were extensively screened and evaluated for their XO inhibitory activity in vitro. Molecular docking and molecular dynamics (MD) simulation were further used to explore the interaction mechanism between flavonoids and XO. The diosmetin, luteolin, and kaempferol were innovatively found to inhibit the UA production of AML12 cells in a time- and dose-dependent manner, in which the IC50 is 6.56, 8.92, and 10.46 μM, respectively. Combined with the MD simulations of the complexes of flavonoids and XO, results showed that flavonoids bind the Mo-molybdopterin domain of XO mainly through hydrogen bonding and hydrophobicity. Lys772 and Thr1011 were the key sites to enhance the inhibitory activity of XO.