The development of bispecific antibodies (BsAbs) has had a profound impact on cancerimmunotherapy. Single-domain antibodies (SdAbs) could offer advantages over otherantibody formats for the generation of a BsAbs, such as small size (12–15 kDa), withhigh affinity and specificity, superior accessibility, and high yield expression in bacteria.In this study, VEGFR2 and CD16 were chosen as the targets to construct BsAbs. As therationale, VEGFR2 is critical for tumor-associated angiogenesis, and CD16 expressed onnatural killer cells is an important target on immune cells. Humanized anti-VEGFR2SdAb 3VGR19 and anti-CD16 SdAb C21 were combined to construct several bispecificSdAbs (BiSdAbs). The biochemical properties of the BiSdAbs were characterized. Theyretained the high affinity for both targets, binding selectivity, and antiangiogenicactivity such as inhibition of cell proliferation, migration, endothelial tube formation,angiogenesis, and cytotoxicity to cancer cells in vitro, indicating that BiSdAbs could bea potential alternative for cancer therapy.