Cancer immunotherapy have changed the paradigm of cancer treatment, but there remains a great need for improvement given that less patients with tumors respond to the treatment of PD-1/PD-L1 blockade. TIGIT (also called T cell immunoreceptor with Ig and ITIM domains), a novel immune checkpoint molecule, has been shown a promising target for drug development of immunotherapy. Here we report generation and characterization of a multivalent bispecific antibody (BsAb) that co-targets PD-L1 and TIGIT. The BsAb consists of tetravalent anti-PD-L1 Fc-fusion nanobody (Nb) and tetravalent anti-TIGIT Nb. The parental anti-PD-L1 Nb showed high specificity and affinity to primate PD-L1, the enhanced T cell activity in vitro and anti-tumor activity in vivo. Similarly, the parental anti-TIGIT Nb showed the high specificity and affinity to primate TIGIT and the enhanced T cell activity. Furthermore, we demonstrated that the BsAb retained high blocking activity towards PD-1/PD-L1 or TIGIT/CD155 interaction. The BsAb synergistically enhanced T cell activities in vitro compared to two parental Nbs. Taken together, we obtained a multivalent BsAb blocking biological function of PD-L1 and TIGIT and it is worthy to further study the anti-tumor activities of this BsAb in vivo. (C) 2020 Elsevier Inc. All rights reserved.