Antioxidant peptides from gelatin hydrolysate of seabass (Lates calcarifer) skin prepared by Alcalase were isolated using a series of chromatographic techniques including Sephadex G‐25 column and reversed‐phase high performance liquid chromatography. Identification of the antioxidant peptides was carried out using UPLC–ESI‐MS/MS. Four potent antioxidant peptides with 5–12 amino acid residues were identified as Gly‐Leu‐Phe‐Gly‐Pro‐Arg (646.3671 Da), Gly‐Ala‐Thr‐Gly‐Pro‐Gln‐Gly‐Pro‐Leu‐Gly‐Pro‐Arg (1,107.5905 Da), Val‐Leu‐Gly‐Pro‐Phe (532.3130 Da), and Gln‐Leu‐Gly‐Pro‐Leu‐Gly‐Pro‐Val (780.4614 Da). The identified peptides were synthesized and analyzed for 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid radical scavenging activity. Gly‐Leu‐Phe‐Gly‐Pro‐Arg exhibited the highest antioxidant activity (81.41 mmol TE/µmol peptide) (P < .05). Therefore, peptides prepared from seabass skin could be employed as functional food ingredients. Practical applications Seabass skin, by‐product from fish processing industry, is a potential raw material for production of protein hydrolysates with bioactivities. Enzymatic cleavage of fish skin gelatin has been used to produce antioxidant peptides. In this study, the potent antioxidant peptides from seabass skin gelatin hydrolysate were purified and identified using a series of chromatographic techniques. Seabass skin gelatin hydrolysate could be used as potential functional food ingredient or nutraceutical with antioxidant activity.