Abstract: To investigate the structure-activity relationship (SAR) of flavonoids from mulberry leaves in inhibiting xanthine oxidase (XOD), this study measured the in vitro inhibitory activities of 17 mulberry leaf flavonoids against XOD to screen for potent compounds. Based on three-dimensional quantitative structure-activity relationship (3D-QSAR) models—comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA)—the SAR underlying their XOD inhibitory activity was analyzed. Molecular docking was employed to simulate the interactions between active compounds and key amino acid residues of XOD. The inhibitory activities of five structurally related compounds against XOD were further determined to validate the predictive reliability of the established models. The results showed that morusin exhibited strong XOD inhibitory activity (IC₅₀=5.65 μmol/L). At20 μmol/L, morusin reduced uric acid levels by 82.27% in a hyperuricemic cell model. 3D-QSAR analysis indicated that the hydrophobic field contributed significantly to the activity. Molecular docking revealed key hydrophobic interactions between active compounds and residues such as PHE-307 and TRP-525 of XOD. The activity trend of the validation set compounds was consistent with model predictions (test set R²>0.6), confirming that the introduction of hydrophobic groups significantly enhanced inhibitory activity. Therefore, the uric acid–lowering activity of mulberry leaf flavonoids was closely related to their hydrophobic field characteristics, with the combined effect of hydrophobic units such as prenyl groups and ortho-diphenolic hydroxyl structures enhancing the inhibitory capacity against XOD.