In order to systematically explore the molecular mechanism of ferulic acid suppressing the activity of xanthine oxidase (XO), the effects of ferulic acid on the inhibitory reversibility, inhibition kinetics, binding properties, and structure were determined by enzymatic and spectroscopic methods. Molecular simulation technology was used to predict the binding conformation of ferulic acid with XO. The results indicated that ferulic acid could reversibly inhibit XO activity in a mixed-type competitive manner with an inhibiting constant of 4.5μmol/L. Ferulic acid had the strongest inhibitory activity on XO with the IC₅₀ of 116.2μmol/L compared to caffeic acid, p-coumaric acid, chlorogenic acid, and gallic acid. Fluorescence titration experiments showed that ferulic acid could quench the fluorescence of XO through static and dynamic quenching procedures, among which static quenching was dominant. XO had a binding site for ferulic acid with a binding constant of 3.38×10⁴L/mol at 298K. The binding process was spontaneously driven by hydrogen bonds and hydrophobic interactions due to ΔG<0, ΔH<0, and ΔS>0. Molecular docking revealed that ferulic acid entered the flavin adenine dinucleotides active center of XO and then affected the normal catalytic process of XO. The analysis of synchronous fluorescence and circular dichroism demonstrated that ferulic acid could alter the secondary and tertiary structure of XO. The polarity around tryptophan and tyrosine residues in XO was enhanced and their hydrophobicity was reduced. The α-helix content was increased and the β-fold content was decreased in XO, making its secondary structure tend to be tight. That was another factor impacting the XO activity. This study could provide a certain theoretical basis for the improvement of hyperuricemia through ferulic acid as an XO inhibitor.