Abstract: Objective To compare the killing effect of anti-CD3/anti-CD133 bispecific antibodies - cytokine-induced killer (BsAb-CIK) with general CIK cells on CD133 positive tumor cells． Methods Hybridoma technique was used to generate human anti-CD133 monoclonal antibody, and enzyme-linked immunosorbent assay (ELISA) was utilized to detect the titer of antibody． Anti-CD3/anti-CD133 bispecific antibodies were produced through chemical coupling with anti-CD3 and anti-CD133 antibody, which was purified by protein G． The killing effects of BsAb-CIK and general CIK on CD133 positive cancer cells were evaluated by CCK-8． Results The titer of anti-CD133 antibody in ascites was 1∶50 000 and the concentration was 3．2 mg/ml． The CIKs bounded by bispecific antibody were established successfully． The experiment results showed that mBsAb-CIK had better activity than general CIK in mediating lysis of CD133 positive cells, such as SW1990, 7901 and A2780 cells, in the same condition of effector-to-target ratio． In effector-to-target ratio of 20∶1, the killing effects was enhanced about 30．99% to SW1990 by BsAb-CIK comparing to general CIK, and it also enhanced about 24．42% to SGC7901 with significant differences (P< 0．05)． Conclusion The killing effects of CIK to CD133 positive tumor cells can be enhanced significantly by anti-CD3/anti-CD133 bispecific antibodies．