Abstract: In this study, using angiotensin converting enzyme (ACE) inhibitory peptides PHP1 and PHP2 as the parent peptides, the hydrophobicity and electrical properties of target polypeptides were altered by substituting amino acid residues. In addition, the potential bioactivity of these polypeptides was assessed using bioinformatics tools. Following this analysis, 19 polypeptide analogues were designed and synthesized using solid-phase synthesis, and their bioactivities were detected in vitro. The results revealed that the polypeptide analogues showed relatively high ACE inhibitory activities, PHP1A-6 (IC₅₀=3.87 μmol/L), PHP2A-3 (IC₅₀=3.33 μmol/L), PHP2A-4 (IC₅₀=2.86 μmol/L), and PHP2A-7 (IC₅₀=4.58 μmol/L) exhibited the highest ACE inhibitory activity levels, significantly higher than the parent peptides (P<0.05). PHP1A-3, PHP1A-4, PHP1A-7, PHP2A-1 and PHP2A-10 displayed equal levels of inhibitory activity in comparison to the parent peptides (IC₅₀<10 μmol/L). Compared with the parent peptides, the α-glucosidase inhibitory activity levels demonstrated by most of the polypeptide analogues were significantly enhanced, PHP1A-3 (IC₅₀=3.09 μmol/L), PHP1A-7 (IC₅₀=9.51 μmol/L), PHP2A-6 (IC₅₀=5.58 μmol/L), PHP2A-11 (IC₅₀=2.35 μmol/L), and PHP2A-12 (IC₅₀=3.98 μmol/L) exhibited the highest activities. Additionally, PHP1A-3 and PHP1A-7 displayed relatively strong inhibitory activity against both ACE and α-glucosidase. At concentrations of 1 mg/mL, the polypeptides containing Cys displayed an ABTS⁺· scavenging rate of higher than 85%, demonstrating a potential antioxidant activity. The structure-activity relationship between the ACE inhibitory peptides and ACE were explored using molecular docking. The results reflected that inhibitory peptides produced multiple stable hydrogen bonds, hydrophobic interactions, π-π stacking interactions, and salt bridges with ACE amino acid residues, thereby improving the inhibitory effects exerted on ACE.