Abstract: Exploring the xanthine oxidase (XOD) inhibitory peptide from Chlorella pyrenoidosa could provide a scientific basis for hyperuricemia prevention and treatment strategies, and promote the comprehensive utilization of microalgal protein resources. In this study, Chlorella pyrenoidosa was used as the raw material to extract proteins. With the XOD inhibition rate and the degree of hydrolysis (DH) as evaluation indicators, the optimal enzymatic hydrolysis conditions were optimized through single-factor and response surface experiments. Based on this, further analysis of XOD inhibitory peptide was conducted. The results showed that papain was the most suitable protease, and the optimal enzymatic hydrolysis conditions were pH7.0, hydrolysis temperature 48.0 ℃, hydrolysis time 4.0 h, enzyme dosage 2000 U/g, and substrate concentration 10 mg/mL. Under these conditions, the theoretical inhibition rate was 73.78%, and the actual inhibition rate reached 71.56%±0.51%. The amino acid composition of Chlorella pyrenoidosa XOD inhibitory peptide was reasonable, with essential amino acids, hydrophobic amino acids, and basic amino acids accounting for 43.17%, 45.07%, and 14.15% of the total, respectively. Additionally, they exhibited moderate stability under gastrointestinal digestion conditions, but their inhibitory activity decreased significantly under high temperature or strong acid/alkaline conditions. They were also relatively sensitive to metal ions such as Fe²⁺, Fe³⁺, Cu²⁺ and Mg²⁺. The relative molecular mass mainly concentrated below 1 kDa, and the ultrafiltered fraction with a molecular weight <3 kDa showed the highest XOD inhibitory activity, with an IC₅₀ of (5.23±0.68) mg/mL. This study provides a theoretical reference for the development and utilization of food-derived uric acid-lowering peptides.