•A novel peptide ATVY demonstrating high scavenging activity to ABTS was purified.•The crucial amino acid as key action site in ATVY was Tyr in N-terminal.•The scavenging kinetics of ATVY on ABTS demonstrated a two-phase reaction process.•ABTS+-Peptide was formed by covalently bonding through phenol group of ATVY to ABTS.•ATVY can be used as a natural agent in controlling food oxidation in food industry. Marine byproducts have become popular research themes due to their biological significance. The black sharkskin protein hydrolysate-based antioxidant peptides were firstly investigated in this study. The black sharkskin-derived novel antioxidant peptide demonstrating 81.05% free radical scavenging activity to ABTS at 500 μg/mL, was identified to be Ala-Thr-Val-Tyr (ATVY). The crucially antioxidant interaction site of ATVY action on ABTS was determined to be Tyr in the N-terminal. ATVY reacted with ABTS to generate polyphenol-derived adducts which subsequently degraded into a purple compound. The MS/MS showed it was formed by covalently bonding through the phenol group of ATVY to the N group of ABTS. The free radical scavenging kinetics of ATVY on ABTS demonstrated a two-phase reaction process. These findings reveal the action mechanism of ATVY on ABTS, implying ATVY can be incorporated in the production of antioxidant food additives.