In this study, the inhibitory activity and mechanism of three gingerols (6-, 8- and 10-gingerol) on xanthine oxidase (XOD), a key enzyme responsible for the formation of uric acid, were evaluated. The three gingerols reversibly inhibited XOD activity in a mixed manner with IC50 values of (13.16 ± 0.41), (10.18 ± 0.22) and (6.88 ± 0.11) μM, respectively. 10-gingerol exhibited stronger inhibitory ability, which might contribute to its superior antioxidant properties, higher binding affinity and more significant conformational changes for XOD compared to other gingerols. Fluorescence quenching and molecular docking results indicated that gingerols acted in the vicinity of the flavin adenine dinucleotide in XOD, interacting with the surrounding key amino acid residues through hydrogen bonding and van der Waals forces, thereby preventing substrate entry and the release of catalytic products. Molecular dynamics simulations showed that the addition of gingerol decreased the stability and increased the structural density of XOD. These findings could provide valuable data for the application of gingerols in new XOD inhibitors and hypouricemic functional foods.