Aim To obtain genetically engineered cytokine with potentialities of clinical application. Methods Anti-human hepatocellular carcinoma (HCC) single chain gene fragment(scFv25) was linked with human TNFα gene to form a fusion gene of anti--HCC cytokine， then it was subcloned into prokaryotic GST fusion expression vector pGEX 4T-1 and expressed in the host E.coli. Indirect immunofluorescent staining was performed on HCC cell smearing slides in order to evaluate the activity of purified aim protein, then initial tumor regression trial was carried out in nude mice bearing HCC to evaluate the targeting therapeutic value of scFv25-TNFα . Results scFv25-TNFα had similar specificity as parental antibody HAb25 for SMMC-7721 antigen. The tumor regression trial to the 2 mm HCC xenografts in nude mice showed that scFv25-TNFα had certain targeting cytotoxicity capacity and the efficiency was 3/3 with complete remission. The cytotoxicity of scFv25-TNFα was better than that of TNFα , whose efficiency was only 2/3 and no complete remission was seen. Conclusion scFv25-TNFα is the anti-HCC cytokine, which have certain potentialities of clinical application.