Based on the alcalase hydrolysis, the grass carp scales gelatin were subjected to two-step enzymatic hydrolysis with trypsin, flavor protease, papain and pepsin respectively, to prepare antioxidant peptides. Results showed that two-step enzymatic hydrolysis could effectively increase the hydrolysis degree and decrease the molecular weight of hydrolysates. The alcalase-trypsin hydrolysates had significant free radical scavenging ability and Fe²⁺ chelating ability. Response surface methodology was performed to optimize the two-step enzymatic hydrolysis conditions, and the optimal enzymatic hydrolysis parameters were as follows: substrate concentration of 100 mg/mL, pH 7.8, hydrolysis temperature of 53 ℃ and hydrolysis time of 50 min. Under this optimal hydrolysis condition, the maximum Fe²⁺ chelating ability was 65.72% and the IC₅₀ value for DPPH·, ·OH and O^-2· were 7.39, 0.68 and 1.84 mg/mL, respectively.