In order to obtain higher added-value of skipjack tuna (Katsuwonus pelamis) swim bladder, compound proteinase was selected for enzymatic hydrolysis of skipjack swim bladder from seven enzymes including bromelaine, compound proteinase, alkaline proteinase, papain, pepsin, trypsin and dispase. With the DPPH radical scavenging rate of hydrolysates as evaluating index, single-factor experiment was employed to investigate the effects of enzyme dosage, initial pH, reaction temperature and time on the antioxidant activity of hydrolysates separately. On the basis of the above observations of single-factor experiment, the optimum conditions for preparing antioxidant hydrolysates of skipjack tuna swim bladder were explored through response surface methodology (RSM). The results showed that, the optimal hydrolysis conditions were as follows: 8.53 U/ mg of enzyme dosage, 5.54 of initial solution pH, 50.03 ℃ of reaction temperature and 5.07 h of reaction time. Furthermore, the hydrolysates were primarily sorted into three fractions with different molecular weight above 10 000 u, 3000 to 10 000 u and 0 to 3000 u by using ultrafiltration. The inhibitory concentration 50% (IC₅₀) values to DPPH radical of three fractions were 0.64, 0.52 and 0.37 mg/mL, respectively. It is demonstrated that DPPH radical scavenging rate of the hydrolysates could be up to 72.00% under these optimal conditions, which was in good agreement with the predicted value of 71.60%, and that fraction below 3000 u of the hydrolysates possesses the strongest effect. Preliminary results indicate that the hydrolysates of skipjack swim bladder could be an effective ingredient for antioxidant products such as health food or cosmetics.