Abstract: To achieve the high-value utilization of squid skin obtained from squid processing, this study utilized an enzymatic approach to prepare protein peptides. The enzymatic hydrolysis parameters were optimized using single factor and response surface tests, with the concentration of peptide and trichloroacetic acid-nitrogen soluble index (TCA-NSI) serving as evaluation criteria. The amino acid composition, molecular weight distribution, peptide sequence, antioxidant activity, and in vitro simulated digestive properties of the prepared protein peptides were also thoroughly characterized. The results showed that the optimal conditions for enzymatic hydrolysis were as follows: Enzymatic temperature of 43.50 ℃, enzymatic time of 100 minutes, enzymatic pH of 7.50, trypsin to alkaline protease ratio of 2:1, and enzyme dosage of 4000 U/g. Under these specified conditions, the TCA-NSI achieved a value of 89.02%±0.66%. The prepared protein peptides exhibited a well-balanced amino acid composition, with an essential amino acid index (EAAI) value of 0.90. Furthermore, the prepared protein peptides encompassed a total of 43 polypeptide sequences, with 38 of these segments exhibiting molecular weights below 1800 Da. The IC₅₀ values for scavenging DPPH radical, ABTS⁺ radical, and hydroxyl radical were determined to be 0.61, 0.28 and 1.95 mg/mL, respectively. Notably, the antioxidant activity remained favorable even after undergoing simulated gastrointestinal digestion in vitro. These findings serve to establish a solid theoretical basis for the advancement and exploitation of byproducts derived from squid processing, as well as their subsequent value-added utilization.