Response surface method (RSM), based on Box-Behnken design, was used to optimize the enzymatic hydrolysis conditions of flatfish skin protein hydrolysates (FSPH). Among the tested proteases, the combination of nutrase and trypsin was selected. The optimal hydrolysis conditions were as follows: pH 7.3, temperature 51.8°C, and the enzyme/substrate (E/S) ratio 2.5; under these conditions, the maximum peptide yield (PY) was 69.41±0.43%. The physiochemical analysis showed that the amino acids (His, Asp and Glu) of FSPH accounted for 18.15%, and FSPH was a mixture of polypeptides mostly distributed among 900–2000 Da. FSPH could exhibit a 93% chelating effect on ferrous ion at a concentration of 400 μg/mL, and also a notable reducing power. This study showed bioprocess for the production of FSPH for the first time, which had a good potential for valuable ingredients in the food, cosmetic and medicine industries.