Objective · To construct an expression vector for human cyclophilin B (hCypB) and prepare active CypB via the prokaryotic expression system. Methods · The DNA sequence enconding hCypB was amplified by PCR and inserted into expression vector pGEX-6P-1 by double enzyme digestion. hCypB was further purified with affinity chromatography and the activity of over-expressed hCypB was verified with GST-pulldown. Results · The recombinant CypB expression plasmid was successfully constructed and verified with DNA sequencing. GST-CypB fusion protein was purified to a purity of 90% by affinity chromatography with a yield of 26 mg of fusion protein from a liter of overnight E. coli culture. GST-pulldown further confirmed that this GST-hCypB fusion protein could form the ternary complex with prolyl 3-hydroxylase 1 (P3H1) and cartilage-associated protein (CRTAP). Conclusion · Large amount of active and highly purified hCypB can be obtained via this experiment method within short time period.