Foodborne xanthine oxidase (XO) inhibitory peptides can decrease the production of uric acid via inhibiting the activity of XO, in order to relieve hyperuricemia. Current researches focused on screening XO inhibitory peptides, but their activities were still need to be improved, and the inhibitory mechanisms were not yet clear. Undaria pinnatifida proteins were hydrolyzed by different proteases, and XO inhibitory peptides were screened by chromatographic fractionation and peptide segment identification, of which inhibitory mechanisms were investigated. The combination of papain and Aspergillus niger acid protease (AnproA1) was the best formulation to hydrolyze Undaria pinnatifida proteins with high XO inhibitory activity (92.1%). The hydrolysate was then purified to identify five novel XO inhibitory peptides, including YLGY, VYW, VVSW, TVVW and KVFAW, in which VYW (IC₅₀=4.3mmol/L) and TVVW (IC₅₀=2.5mmol/L) showed high XO inhibitory activities. The tryptophan residues on VYW and TVVW formed Pi-Pi stacking and Pi-Alkyl interactions with the active center of XO and other sites, showing non-competitive and competitive inhibitor effect, respectively. XO inhibitory peptides from Undaria pinnatifida protein were prepared by the combination of papain and AnproA1, which would be theoretically and practically important to develop anti-hyperuricemic food ingredients.