The invention provides an amphiphilic alpha-helical polypeptide for inhibiting PD-L1 (programmed death-ligand 1) and application of the amphiphilic alpha-helical polypeptide. By investigating the in-vitro PD-1/PD-L1 protein binding inhibition effect, the in-vivo and in-vitro anti-tumor effect and the in-vivo immune activation effect of the polypeptide, it is verified that the polypeptide can block a PD-1/PD-L1 signal channel and activate the immune response of T cells and has the anti-tumor effect. The polypeptide disclosed by the invention can be prepared by adopting a solid-phase synthesis method, and compared with a monoclonal antibody and a small molecule compound, the polypeptide disclosed by the invention solves the problems of insufficient immune-related toxicity, pharmacokinetics and tumor permeation, high cost burden of manufacturers and consumers and the like.