〔Abstract〕 Objective To express human glucose 6-phosphate dehydrogenase(G6 PD) and establish its inhibitor screening modelin vitro. Methods G6 PD cDNA was amplified by PCR with a couple of primers flanked withNdeI andXhoI site at their 5′ terminals, respectively. G6 PD cDNA and pET-28 a plasmids were digested byNdeI andXhoI, and then linked. The linked products were transformed into Escherichia coli(E.coli)Top10. Recombinant plasmids were identified and transformed intoE.coliBL21(DE3), the expression of G6 PD was induced by 0.4 mmol/L IPTG, recombinant G6 PD was purified by Ni2+affinity column. Glucose 6-phosphate and NADP+ were used as substrate and coenzyme of G6 PD, respectively. G6 PD's activity was observed by the shift of A340of NADPH. The model was optimized to give the greatest performing concentration of G6 PD and dehydroepiandrosterone(G6 PD's inhibitor). Results Recombinant plasmid pET-28 a-G6 PD was constructed, soluble G6 PD was expressed in BL21(DE3) with a yield of 2.5 mg/Lviathe induction of IPTG. The optimum G6 PD concentration was 900 μg/L. The high Z′ score of 0.88 was achieved using 20 μmol/L DHEA as positive control. Conclusion Active G6 PD is expressed inE.coli, and this assay is highly suitable for screening of G6 PD inhibitorin vitro.