Abstract: Background The Pacific abalone (Haliotis discus hannai) is one of the predominant aquaculture species in Fujian Province, China. During processing, visceral by-products—accounting for 15–25% of live weight—are typically discarded, leading to significant resource wastage and environmental concerns. The extraction of bioactive peptides from these underutilized by-products represents a critical pathway for advancing value-added processing industries while addressing sustainability challenges. This approach not only mitigates resource inefficiency but also aligns with escalating demands for health-oriented functional products. Notably, the derived peptides exhibit antioxidant, anti-inflammatory, and antitumor properties, which have garnered significant attention for their potential applications in functional food development and pharmaceutical innovation. Objective To address the demand for high-value utilization of abalone visceral processing by-products, this study focuses on xanthine oxidase (XOD), a critical therapeutic target in hyperuricemia management, to establish an XOD-inhibitory peptide screening system. By optimizing enzymatic hydrolysis conditions for abalone viscera, the research refines the bioactive peptide screening framework for abalone by-products, elucidates the relationship between key processing parameters and product bioactivity, and provides foundational data to advance industrial-scale development. These efforts aim to bridge the gap between sustainable resource utilization and the production of functional ingredients with therapeutic applications. Methods A combined strategy integrating single-factor experiments and response surface methodology was employed to systematically investigate the influence of enzymatic hydrolysis parameters on bioactive peptide preparation from abalone viscera. Leveraging the high-efficiency proteolytic properties of alkaline protease, dual evaluation metrics—degree of hydrolysis (DH) and xanthine oxidase (XOD) inhibitory rate—were utilized for process assessment. A process optimization model was developed using Box-Behnken design (BBD), with parameter interactions and their correlations to bioactivity elucidated through three-dimensional response surfaces and contour plot analyses. This approach quantitatively delineates the synergistic effects of enzymatic variables on functional outcomes, providing a robust framework for scalable bioactive peptide production. Results ‌XOD Detection System Development:‌ Substrate-enzyme kinetic experiments were conducted to establish stable reaction conditions, including a substrate concentration of 0.24 mmol/L, XOD concentration of 20 μg/mL, pH 7.5, reaction temperature of 25°C, and reaction duration of 30 min. ‌Enzymatic Hydrolysis Process Optimization:‌ The optimal parameter combination was determined as a solid-to-liquid ratio of 1:50, enzyme dosage of 4000 U/g, temperature of 61°C, pH 9, and hydrolysis time of 4 h. The optimized hydrolysate exhibited an XOD inhibition rate of 89.70%±1.23% and a degree of hydrolysis of 20.34%±0.85%. Significance‌ The integrated system combining XOD activity assay and enzymatic hydrolysis process optimization achieves the first targeted preparation of XOD-inhibitory peptides from abalone viscera. This methodology advances the development of bioactive peptides from marine processing by-products and demonstrates significant practical value in promoting resource utilization of abalone-derived waste. The framework establishes a novel approach for precision enzyme-driven bioactive compound extraction, bridging functional ingredient discovery with sustainable valorization of underutilized marine biomass.