Hyperuricemia is characterized by elevated uric acid (UA) level in the body. The xanthine oxidase (XO) inhibitory ability is an important way to evaluate the anti-hyperuricemia effect of natural products. Ferulic acid (FA) is a phenolic acid compound, and it is a free radical scavenger with many physiological functions. The aim of this study was to investigate the structure-activity relationship, potential mechanism and interaction of FA as XO's inhibitor. In the cell experiment, using 1.25 mM adenosine to incubate for 24 h under the optimal conditions (37 °C, pH = 7.2) can increase the UA production by 1.34 folds. PCR analysis showed that FA could reduce the mRNA expression level of XO. FA inhibited XO in a mixed mode (IC50 = 13.25 μM). The fluorescence quenching of XO by FA occurs through a static mechanism, with an inhibition constant of Ki = 9.527 × 10−5 mol L−1 and an apparent coefficient of α = 1.768. The enthalpy and entropy changes were found as −267.79 KJ mol−1 and − 860.85 KJ mol−1, indicating that both hydrogen binding and hydrophobic are involved in the interaction of this polyphenolic natural compound with XO. Thus, FA supplementation may be a potential therapeutic strategy to improve hyperuricemia by reducing UA production.