Abstract: In this paper, the active parts of sea cucumber (Apostichopus japonicus) with high antihypertensive activity were screened and the preparation process of active peptides was optimized. Different parts (body wall, intestine, and ovum) of A. japonicus were hydrolyzed by enzymolysis, and the ACE inhibition rate was used as an indicator to screen the optimal protease. The optimal active site for inhibition was selected by comparative screening of the half maximal inhibitory concentration (IC₅₀) determination of ACE inhibitory rate of each lysate. Single factor and response surface tests were used to determine the optimum enzymatic hydrolysis conditions of the active peptides. The relative molecular weight of the protease hydrolysates was determined to determine its distribution range. The ACE inhibition activity of different components was analyzed after separation by ultrafiltration membrane. Search results, alkaline protease was selected as the optimal hydrolytic enzyme, and the IC₅₀ values of ACE inhibition of each protease lysate from body wall, intestine and ovum were 1.11, 4.02, 0.65 mg/mL, respectively, so that A. japonicus ovum had a better ACE inhibition effect and were the optimal active site for inhibition. Its optimal preparation process parameters for enzymatic hydrolysis were as follows: 5 h enzymatic hydrolysis time, 3.5 U/mg enzyme added, 65.26 °C enzymatic hydrolysis temperature, 3.51% substrate concentration, pH9.02 enzymatic hydrolysis, and ACE inhibition rate of A. japonicusr ovum was 80.65%±0.52% under these conditions, which was close to the predicted value. The molecular weight of proteolytic products was concentrated under 3000 Da, accounting for 98.37% of the total content, of which 1000~3000 Da accounted for 9.50%, and less than 1000 Da accounted for 88.87%. The ACE inhibitory activity of oligopeptide components (IC₅₀=0.30 mg/mL) isolated by ultrafiltration membrane was significantly higher than that of hydrolysates and trapped liquid components after process optimization. The results of this study would provide a theoretical basis for high-quality utilization of the by-products of A. japonicus, which could be used as high-quality resources for the isolation and purification of antihypertensive peptides.