Our aim was to examine the uric acid-lowering potential of mulberry (Fructus mori) aqueous extract and its key bioactive constituents. A zebrafish model of acute hyperuricemia was induced by combined treatment with potassium oxonate (200 μmol/L) and sodium xanthine (10 μmol/L). After intervention with mulberry aqueous extract at varying concentrations (2.5, 5, and 10 mg/L), uric acid levels, xanthine oxidase (XOD) activity, and adenosine deaminase (ADA) activity were quantified in zebrafish. The findings demonstrated that mulberry aqueous extract significantly lowered uric acid levels by suppressing XOD activity. Further analysis of bioactive components in the extract was performed using high-performance liquid chromatography-mass spectrometry (HPLC-MS), followed by molecular docking with AutoDock Vina, targeting XOD as the ligand. Based on docking activation energies, six compounds with notable XOD inhibitory activity were identified and their in vitro XOD inhibitory activity was ranked in the decreasing order of quercetin, mulberrin, astragalin, escin, caffeic acid, and protocatechuic acid. In summary, mulberry aqueous extract exhibits a pronounced uric acid-lowering effect, likely mediated by the XOD inhibitory activity of its bioactive compounds (quercetin, mulberrin, astragalin, escin, caffeic acid, and protocatechuic acid).