Objective： This study aimed to extract polypeptide from the castoff of Thunnus alalunga and explore its protective effects on the damaged Chang liver cells induced by H2O2. Methods： The castoff of T. alalungawas pretreated for enzymatic hydrolysis. The optimal enzyme was screened using relative growth rate as the indicator. The optimal enzymolysis condition was determined by orthogonal tests. After hydrolysis， the resulting product was orderly purified by the ultrafiltration， ion exchange chromatography， gel filtration chromatography and reversed-phase high-performance liquid chromatography， and MTT method was used to evaluate the purification capacity for each step. The activities of AST and ALT enzymes were assayed by kit according to the manufacturer’s instruction. Inverted microscope and the flow cytometric analysis observation were used to investigate the effects of polypeptide addition on the apoptosis of Chang liver cells induced by H2O2. Results： The optimal enzyme for enzymolysis was trypsin， and its corresponding optimal hydrolysis condition was determined as follow： solid-liquid ratio was 1∶3， pH value was 9， enzyme dose was 900 U/g， temperature was maintained at 45 ℃ and held for 5 h. After a series of purification， pure polypeptide was harvested and its amino acid sequence was identified as Gly-Ala-Pro-Gly-Glu-Arg-Gly-Ser-Lys-Cys-Phe-Lys. With the addition of the identified polypeptide on Chang liver cells， the activities of ALT， AST， ADH and MDA were decreased but the activities of SOD were increased relative to control group without polypeptide during the period of H2O2 induction. The flow cytometry results demonstrated that the late apoptotic cell amount was significantly decreased relative to the control group. Conclusion： The identified T. alalunga polypeptide had a protective effect on the H2O2 induced Chang liver.