Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells (PBMCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eukaryotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detected by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1.RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P < 0.01) after transfection in THP-1 cells. Conclusions We successfully constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foundation for further study on IL-37 functions and its association with related diseases.