► Peptide with high antioxidant properties was isolated from the visceral waste of Magalaspis cordyla. ► Two steps of purification were executed using FPLC to obtain the active peptide. ► The sequence of purified peptide was determined as Ala–Cys–Phe–Leu (518.5Da). ► Identified peptide proved better than natural antioxidant, α-tocopherol in PUFA protection. In the present study, a peptide having high antioxidant properties was isolated from horse mackerel viscera protein, Magalaspis cordyla. In vitro gastrointestinal digestion was employed to obtain potential protein hydrolysate and was subjected to consecutive chromatographic methods using fast protein liquid chromatography (FPLC) connected to diethyl amino ethyl (DEAE) anion exchange column and Sephadex G-25 gel filtration column. The activity of the fractions was tested against DPPH and hydroxyl radicals and the isolated peptide showed 89.2 and 59.1 percentage of scavenging. The amino acid sequence of purified peptide was determined using ESI-MS/MS as Ala–Cys–Phe–Leu (518.5Da), it exhibited high activity against polyunsaturated fatty acid (PUFA) peroxidation than that of natural antioxidant, α-tocopherol.