Abstract: Blocking the interaction of PD-1/PD-L1, which has a preferable therapeutic effect on malignant tumors, can activate tumor-infiltrating T cells and restore their anti-tumor activities. Although antibody drugs targeting the PD-1/PD-L1 pathway have shown certain efficacy in treating cancer patients, existing antibody drugs have problems such as high production costs, large individual differences, and inducing inappropriate immune responses and unavoidable defects such as poor organ or tumor permeability, inferior oral bioavailability, etc. Therefore, peptide inhibitors are urgently needed to make up for the shortcomings of current PD-1/PD-L1 interaction antibody blockers. In this study, we took the recombinant human PD-1 protein as the target and used next-generation phage display to pan the Ph.D.-7 and Ph.D.-12 phage display libraries for the first time, and isolated 400 potential PD-1 binding peptides. We then clustered the PD-1 binding peptide dataset based on the BLOSUM62 scoring matrix. Finally, amino acid composition, position-based amino acid preference and mimotope analysis were performed on the PD-1 binding peptide dataset. The PD-1 binding peptides obtained by NGPD biopanning in this study are expected to be developed into candidate peptide drugs that block the interaction of PD-1/PD-L1. The research results have certain guiding significance for computationally designing PD-1 binding peptides.