Polypeptides from sea cucumber cooking water were prepared by neutral protease, pepsin and trypsin,and the antioxidant activity of the polypeptides was compared with synthetic antioxidant TBHQ by measuring the scavenging capacities to DPPH•,•OH and O-2•,and the relative molecular mass was assayed. The results showed that scavenging capacities of TBHQ and polypeptides prepared by three proteases on free radicals were enhanced with the increase of sample concentration. The corresponding IC50 values of polypeptide from sea cucumber cooking water prepared by neutral protease to •OH，O-2•and DPPH• free radicals were 8.565 mg/ml, 6.658 mg/ml and 2.015 mg/ml respectively, and it had better scavenging ability to O-2•than TBHQ.The molecular weight of polypeptides from sea cucumber cooking water prepared by neutral protease, pepsin and trypsin was respectively less than 2 500,3 500 and 1 000D， analyzed by liquid chromatography.