Introduction <p>Immune checkpoint inhibition, especially suppressing the interaction between programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) to prevent immune evasion, is an important strategy for tumor immunotherapy.</p> Methods <p>Hence, a library of peptide candidates, focusing on the surface features of the PD-1 ligand-binding domain (LBD) and targeting the canyon area (region T1) on PD-1 LBD surface, was developed for immune checkpoint inhibition. The peptides were screened by molecular simulation. The inhibition performance of peptide candidate was further evaluated using cytotoxicity assays, fluorescence microscopy, flow cytometry, T cell proliferation and apoptosis tests, and isotherm analysis.</p> Results <p>Among the peptide candidates, sixteen peptides exhibited sufficient affinity and further molecular simulations indicated that only WY6 (WRWGVY) showed a strong affinity to the region T1 but had little affinity to the cell membrane. The further umbrella sampling (US) simulations revealed a significant increase in the binding potential of WY6 to PD-1 LBD. The cytotoxicity experiments demonstrated that the co-incubation of cells with 100 µM WY6 did not cause significantly decrease in cell viability. The fluorescence microscopy observations indicated that 1 µM WY6 could effectively bind to PD-1 LBD within 1&#xa0;h. The flow cytometry analysis illustrated that the binding of PD-L1 to PD-1 was significantly inhibited (62.6%) in the presence of 100 µM WY6 in fetal bovine serum (FBS), and subsequent cell apoptosis studies showed a T cell apoptosis rate of 12.2%, suggesting that WY6 has favorable ligand characteristics.</p> Conclusion <p>This research proposes a potential immunotherapy drug and a novel strategy for the design of peptide inhibitors.</p>