The B7-H4 molecule, a unique negative regulator of T lymphocytes which is overexpressed on the surface of various tumorcells, is a particularly important target candidate for tumor therapy because it can be blocked with anti-B7-H4 antibodies toinhibit the B7-H4 signaling pathway. Our previous work established an anti-B7-H4 single-chain variable fragment (scFv)library, so we have now amplified the genes encoding anti-B7-H4-scFv and human IgG1 CH3 and ligated them by overlapextension PCR to obtain a recombinant gene. After sequencing, the gene was cloned into the expression vector pET43.1aand expression was induced in E. coli BL21 (DE3) by isopropyl-b-D-1-thiogalactopyranoside (IPTG). The protein waspurified on a nickel-nitrilotriacetic acid (Ni-NTA) resin column and its antigen specificity and affinity were examined byELISA and western blotting. We also established a Lewis lung cancer model in C57BL/6 mice to further identify thebiological function of the scFv protein in vivo. The results showed that tumor volume, body weight and necrotic tissues inthe control group were significantly greater than in the experimental group, indicating that selected scFvs had goodbiological activity and could inhibit tumor growth in tumor-bearing mice. Our work thus offers a new approach for thedevelopment of cancer-targeted therapy.