In this investigation, a DNA sequence was designed and synthesized by joining coding regions of soluble programmed cell death-1 and T-cell immunoglobulin and mucin-3 domains and cloned into the lentiviral expression vector pLVX-IRES-ZsGreen1. After infection of the recombinant lentivirus into HEK 293T cells, the recombinant fusion protein, designated as sP1T3, was successfully expressed as detected by Western blotting. Binding assay was performed by incubating the cell lysate with cancer cells, which have high expressional levels of PD-L1/2 and galectin 9 and subsequent fluorescent staining showed strong membranous signals compared to negative controls using cell lysate without sP1T3, suggesting specific binding of sP1T3 to these ligand-expressing cancer cells. The recombinant fusion protein sP1T3 provided a useful tool for studying the effect of dual-blockade of immune checkpoint and its potential in targeted immunotherapy of cancer, which needs further investigation.