Xanthine oxidase (XO) plays a crucial role in purine metabolism, catalyzing the oxidation of hypoxanthine to xanthine and subsequently to uric acid. Elevated uric acid levels can lead to hyperuricemia, a condition linked to gout, kidney stones, and other chronic diseases. Inhibiting XO activity represents a promising strategy for managing hyperuricemia. This study investigated the inhibitory effects of the flavonoid hesperetin enriched in citrus fruits on XO activity, its antioxidant properties against reactive oxygen species (ROS) generated by the XO reaction, and the underlying mechanisms of enzyme inhibition. Enzyme kinetics and spectroscopy revealed that hesperetin competitively inhibited XO at an inhibition constant of (2.15 ± 0.05) × 10