Abstract: Single-factor and orthogonal experiments were employed to optimize the preparation process of collagen polypeptide through the hydrolysis of the fish collagen by pepsin.The angiotensin converting enzyme ( ACE) inhibitory activity of marine and freshwater fish collagen was determined by high performance liquid chromatography to characterize its blood pressure-lowering function.It showed that the optimal hydrolysis condition of pepsin was emzyme-to-substrate ratio of 1 ∶ 125, substrate concentration 3%, temperature 37 ℃, initial p H2.5 and reaction time 12 h while DH was 19.17%.The results showed that the supernatant of enzymatic hydrolysis of marine fish collagen and freshwater fish collagen possessed higher ACE inhibitory activity, while the IC50 of their hydrolysates to ACE were 1.34 mg/m L and 1.52 mg/m L. The hydrolysate was separated by Amicon Ultra-4 Centrifugal Filter Devices with the molecular weight lower than 3 ku. The inhibitory activities of the two fractions was determined.The results showed that the IC50 of CP-Ⅰ ( ≤3 ku) and CP-Ⅲ ( ≤3 ku) were 0.43 mg/m L and0.60 mg/m L. While the IC50 of CP-Ⅱ ( ≥3 ku) and CP-Ⅳ ( ≥3 ku) were 1.78 mg/m L and 1.69 mg/m L.