Purification of peptides responsible for angiotensin I-converting enzyme (ACE) inhibitory activity from highly complex protein hydrolysates is difficult. Affinity chromatography is a powerful method for purification of peptides. In this study, a metal affinity-immobilized magnetic liposome (MA-IML) was prepared using lipid, N -hexadecyl iminodiacetic acid (HIDA) and magnetic nanoparticles made of FeCl 3 ·6H 2 O and FeCl 2 ·4H 2 O as main materials. MA-IML was used to adsorb ACE inhibitory peptides from lizard fish proteins hydrolysates. The optimal pH of adsorption solution was 8.5. The peptide sample adsorbed by MA-IML was separated by reverse phase-high performance liquid chromatography (RP-HPLC). Upon amino acid sequence analysis and verification, an ACE inhibitory peptide with IC 50 value of 108 μM was identified to be VYP. Molecular docking results indicated that VYP bound to ACE via multiple binding sites. The present study demonstrated that MA-IML might be a useful tool for separating ACE inhibitory peptides from proteins hydrolysates. • MA-IML was prepared and applied in separation of ACE inhibitory peptide from natural product. • The process of peptide separation and the batch recovery of MA-IML could be facilitated with the aid of a magnet simply. • An ACE inhibitory peptide VYP with IC 50 value of 108 µM was purified from lizard fish proteins hydrolysates. • VYP bound to ACE via multiple binding sites, of which the Val made the greatest contribution. [ABSTRACT FROM AUTHOR]