Blockade of the PD-1/PD-L1 axis using antibody drugs has been a clinically efficacious immunotherapy in cancer treatment. However, studies on peptide inhibitors blocking the interaction between PD-1/PD-L1 in cancer treatment in clinical practice have not yet been reported. In this study, a series of peptide inhibitors were synthesized based on a continuous sequence of 14 amino acids from PD-L1 and suitable modifications to form a hairpin structure. The effect of inhibitors on the blockage of PD-1/PD-L1 by increasing the stability of the hairpin structure was determined using BLI and co-culture models. The results showed that increasing the stability of the hairpin improved the affinity of inhibitors to PD-1 and increased IL-2 secretion. Therefore, modifying the hairpin structure of peptide inhibitors may be a useful approach to block the interaction between PD-1 and PD-L1. •P1.1 was examined.•Stabilizing the hairpin increased the affinity to PD-1 and IL-2 secretion, while decreasing the stability does not.•Compared to P1.1, decreasing the stability of the hairpin inhibited the affinity of inhibitors to PD-1 and IL-2 secretion.