The invention discloses a PD-1 targeting fusion protein and related application thereof. A fusion protein preparation capable of effectively targeting and blocking PD-1 molecules is successfully designed and prepared in the invention. The fusion protein preparation shows specific PD-1 binding activity and also can enhance proliferative activity and activation level of T cells. The fusion protein and a PD-1 ligand can competitively bind to a PD-1 receptor. By blocking immunosuppressive signals, immune cellular function is enhanced. The fusion protein has a good application prospect in natural immunity and adaptive immunity mediated tumor immunotherapy.