Immune checkpoint inhibitors represented by PD-1/PD-L1 monoclonal antibodies have shown great success in tumor immunotherapy. However, the response rate of immune checkpoint blockade (ICB) therapy alone is far from satisfactory due to insufficient and exhausted tumor-infiltrating T cells. Meanwhile, antibody-based drugs have some drawbacks such as high cost and complicated preparation, which require further development of nonantibody immune checkpoint inhibitors and more rational strategies for improving the effectiveness of tumor treatment. Here, a highly efficient bifunctional peptide (Bi-pep) was constructed for tumor treatment by simultaneously activating T cells and blocking the PD-L1 immune checkpoint. This peptide not only can block the PD-1/PD-L1 immunosuppressive pathway but also directly and efficiently promote the activation and proliferation of T cells, thereby showing a significant effect on promoting T cell killing of tumor cells. The Bi-pep-induced antitumor effect was verified on both subcutaneous and orthotopic tumor models, which can significantly inhibit tumor growth and thus prolong the survival of tumor-bearing mice, holding great potential for biomedical applications.