Recent studies have shown that PD-1/PD-L1 checkpoint blockade is a dramatic therapy for melanoma through enhancing antitumor immune activity. Currently, major strategies for PD-1/PD-L1 blockade have mainly focused on the use of antibodies and compounds. For seeking an alternative approach, it is of interest to employ endogenous proteins as blocking agents. Considering the facts that extracellular domain of PD-1 (ePD1) includes the specific binding site with PD-L1, we constructed a PD-1-based recombinantly tailored fusion protein (dFv-ePD1) which consists of bivalent variable fragment (dFv) of MMP-2/9-targeted antibody and ePD1. Furthermore, the melanoma-binding intensity and antitumor activity were investigated. As shown, intense and selective binding capability of the protein dFv-ePD1 to human melanoma specimens was confirmed by tissue microarray. In addition, dFv-ePD1 suppressed significantly the migration and invasion of mouse melanoma cells B16-F1 and displayed the cytotoxicity to cancer cells in vitro. Notably, dFv-ePD1 significantly inhibited the growth of mouse melanoma B16-F1 tumor in mice and in vivo fluorescence imaging showed that dFv-ePD was gradually accumulated in B16-F1 tumor, whose fluorescence intensity at tumor site was stronger than that of dFv. The study indicates that the recombinant protein dFv-ePD1 have an intensive melanoma-binding capability and exert potent therapeutic efficacy against melanoma. The novel format of PD-L1-blocked agent may play an active role in antitumor immunotherapy.