The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a LAG‑3 affinity peptide N13, a preparation method and an antitumor application thereof. The affinity peptide includes 12 amino acids, the molecular weight is 1634.8 Da, and its sequence is DDFRVWWPNFPR. The peptide can be prepared by the Fmoc solid-phase polypeptide synthesis method, and at the same time, the peptide can be used as a drug in tumor treatment. In this application, the eukaryotic protein rhLAG3-Fc is used as the target molecule, hIgG1-Fc is used as the tag protein, and the affinity peptide N13 of the extracellular segment of LAG-3 is screened out through the high-throughput technology of phage display peptide library. For this peptide, further in vitro signal pathway blocking experiments and mouse tumor-bearing experiments showed that this peptide has good application prospects in tumor treatment and prolonging the survival of organisms, and provides a new method for the immunotherapy of organism tumors. possible.