Xanthine oxidase (XO) is a purine catabolic enzyme related to hyperuricemia and gout. Porphyra polysaccharide (PP) is a kind of sulfated polysaccharide with potent biological activity. Herein, the interaction mechanism between PP and XO was studied by enzyme kinetics and multi-spectroscopy methods for the first time. Inhibition kinetics assay showed that PP reversibly inhibited XO activity in a mixed competitive manner with an IC50 of 10.53 ± 0.69 mg/ml. Fluorescence titration studies and thermodynamic parameter calculations revealed that PP could spontaneously bind to XO through hydrophobic interactions, with a class of binding site. Circular dichroism analysis demonstrated that PP induced secondary structure rearrangement and conformational change of XO. Molecular docking further revealed that PP inserted into the hydrophobic cavity of XO, occupying the catalytic center, leading to the inhibition of XO activity. This study may provide new insights into the inhibitory mechanism of PP as a promising XO inhibitor.