The antioxidant activities of tuna head protein hydrolysate (THPH) prepared with Alcalase were evaluated. THPH showed evident radical scavenging activity in a dose-dependent manner with the IC50 values for 1,1-diphenyl-2-pycrylhydrazyl (DPPH), superoxide and hydroxyl radicals being 1.34, 1.20 and 2.84 mg mL−1, respectively, and its reducing power was 0.948 at 12.5 mg mL−1. THPH showed good inhibitory activity in soybean oil peroxidation after accelerated oxidation at 60 °C, and the oils with 0.01%, 0.05% and 0.1% THPH had significantly (P < 0.05) lower peroxide values than the control, after storage at 60 °C. Moreover, the inhibited oxidation effect of 0.1% THPH was similar to that of 0.01% butylated hydroxytoluene (BHT). The molecular weight distribution of THPH revealed that 70.5% of the total amount was peptides with molecular weight lower than 5000 Da, composing mostly of low molecular weight peptides located at 1020–2585 Da (30.78%) and 241–1020 Da (37.15%).