In anti-tumor immunotherapy, the dysfunction of immune killer cells responsible for directly killing tumor cells, such as cytotoxic T lymphocytes(CTL), is the direct cause of the failure of immunotherapy. Antigen-specific CD8⁺ T lymphocytes transform into exhausted T cells when the host immune response fails and antigens are present for a long time. The exhaustion of T cells in the tumor may be related to the abundant transforming growth factor-β (TGF-β) in the tumor microenvironment. There have been several anti-tumor immunological strategies targeting TGF-β, and these TGF-β-targeting therapeutic strategies show good anti-tumor activities, which may be partially related to the improvement of T cell exhaustion. T cell exhaustion also occurs during chronic viral infection, and co-expression of inhibitory receptors is a key feature of exhausted CD8⁺ T cells. Here we reviewed the potential relationships and mechanisms between TGF-β and CTL exhaustion in tumor microenvironment and chronic viral infection.