AbstractBispecific antibody (BsAb) has been proved to be a very effective antitumor approach because of its distinctive advantages of immune‐mediated cytotoxicity. To enhance the ability to recruit and activate T lymphocytes for tumor‐specific killing, we constructed and prepared a recombinant human single‐chain Fv bispecific antibody (BsAb), named VEGFR1/CD3 BsAb, targeting VEGFR1 and CD3. The VEGFR1/CD3 BsAb was expressed in CHO‐K1 cells and purified by Ni‐NTA affinity chromatography. The CD3 and VEGFR1‐binding activity of VEGFR1/CD3 BsAb was confirmed by flow cytometry. T lymphocyte activation and proliferation induced by VEGFR1/CD3 BsAb were also demonstrated in vitro. Notably, our VEGFR1/CD3 BsAb presented a powerful and specific killing effect against VEGFR1‐positive human breast cancer cell MDA‐MB‐231 and MDA‐MB‐435 through activating T lymphocyte at very low concentrations, indicating that it will be a valuable antibody drug for treatment of VEGFR1‐positive cancers in the future.