Anti-tumour BsAb (bi-specific antibody) has been proved very effective in killing tumour cells both in vitro and in vivo. In order to enhance its ability to recruit and activate T-lymphocytes and then improve tumour-specific cytolysis, an anti-ovarian carcinoma/CD3 BsAb, BHL-I, was fused to N-terminal 18 peptide of CCL21 (CC chemokine ligand 21) to produce a new chemotactic BsAb, named 18TBHL. It was expressed in soluble form in the cytoplasm of Escherichia coli and purified with DEAE anion-exchange chromatography and immobilized-metal-ion affinity chromatography. The chemotactic ability of 18TBHL to PBLs (peripheral-blood lymphocytes) was detected by Boyden chamber chemotaxis assay. The specific ability to bind to ovarian carcinoma cells, SKOV3, and PBLs was tested by ELISA and flow cytometry. Just as expected, the enhanced tumour-specific cytolysis of 18TBHL was validated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] method and flow cytometry. The results indicated that fusion of chemotactic peptide to BsAb potentiated its cytotoxicity to tumour cells in vitro. It suggests that 18TBHL may be a promising candidate agent in cancer immunotherapy.