This study aimed to investigate the inhibitory effect and mechanism of alginate on xanthine oxidase (XO), and to evaluate its potential as a natural inhibitor or functional food supplement for alleviating hyperuricemia. Sodium alginate (SA) was used as a model compound. Enzymatic kinetics, fluorescence spectroscopy, time-resolved fluorescence spectroscopy (TRFS) and circular dichroism (CD) spectroscopy were employed for comprehensive analysis. The enzymatic kinetics results indicated that SA inhibited XO activity in a reversible mixed-type manner with an inhibition rate of 45.42% at a concentration of 10 mg/mL. Fluorescence spectroscopy analysis revealed that SA reduced the intrinsic fluorescence intensity of XO through a static quenching mechanism, with the interaction occurring at a single binding site. TRFS analysis further confirmed the static quenching process. CD spectroscopy demonstrated that SA induced secondary structural and conformational alterations in XO. The findings suggest that SA effectively inhibits XO activity by binding to its active site and inducing conformational changes in the enzyme, providing a theoretical foundation for the development of related functional foods or therapeutic agents.