The present invention provides a peptide having high binding affinity for PD-L1 protein and use thereof. The peptide has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, or the peptide is a tandem or branched peptide with a single repeat or multiple repeats of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4 and has an amino acid sequence of SEQ ID NO: 5. The peptide can bind to human PD-L1 with high affinity, competitively block the affinity of PD-1/PD-L1 protein, block the negative regulatory tolerance pathway of human tumors, activate immunity and increase the lethality of T cells against tumor cells.