Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.