Plant-derived bioactive peptides have drawn much attention because of their physiological functions. This study aimed to evaluate bioactive peptides in rapeseed protein and identify novel angiotensin Ⅰ-converting enzyme (ACE) inhibitory peptides using bioinformatics methods. A total of 24 kinds of bioactive peptides were encrypted in the 12 selected rapeseed proteins by analysis in BIOPEP-UWM, with higher occurrence frequency of dipeptidyl peptidase Ⅳ (DPP-Ⅳ) inhibitory peptides (0.5727-0.7487) and ACE inhibitory peptides (0.3500-0.5364). Novel ACE inhibitory peptides FQW, FRW and CPF were identified by in silico proteolysis, and they had strong inhibitory effects on ACE in vitro, showing IC50 values of 44.84±1.48μM, 46.30±1.39μM and 131.35±3.87μM, respectively. Molecular docking results displayed that these three peptides were able to interact with ACE active site via hydrogen bonds and hydrophobic interactions, and coordinate with Zn2+. It suggested that rapeseed protein could be a good source for the production of ACE inhibitory peptides.