TIGIT, a T-cell immunoreceptor with Ig and ITIM domains, has been recognized as a critical inhibitory receptor on T cells and natural killer (NK) cells. It plays an important role in immune suppression mediated by tumors. Tumor cells express CD155, which engages TIGIT and hampers T cell or NK cell activation to perform cytotoxicity, thus facilitating tumor progression. Releasing TIGIT on immune cells from CD155 binding to target tumor cells is one promising strategy for cancer immunotherapy. To this end, TIGIT-specific nanobodies were generated by immunizing camels with the TIGIT protein, from which a library of nanobodies was built. Then, Monoclonal nanobodies were screened by phage display, and high-affinity nanobodies for TIGIT were identified. Competitive ELISA assays were performed to demonstrate that nanobodies effectively inhibited the binding of TIGIT to CD155. Furthermore, the cytotoxicity activities of NK-92MI cells were significantly enhanced by adding TIGIT antibodies; meanwhile, higher cell degranulation was determined. At the same time, the viability of tumor cells after mixing with NK-92MI cells was detected, showing that the viabilities of K562 cells were as high as 90%, suggesting the limited cytotoxicity of NK-92MI cells. In contrast, the viabilities of K562 cells were significantly reduced when the nanobodies were added, indicating that nanobodies effectively block TIGIT on NK cells from binding to CD155 on tumor cells. These findings suggest that the screened nanobodies have promising potential for further evaluation in tumor-bearing mouse models. These nanobodies might offer significant benefits in clinical settings by improving patient selection and therapeutic outcomes in cancer immunotherapy.