Objective: To construct the recombinant strain of efficient NADPH regeneration and investigate its optimal process condition. Methods: The key enzyme genes glk and zwf were introduced into Escherichia coli by genetic engineering to achieve efficient NADPH regeneration. The NADPH regeneration condition was explored. Results: The recombinant E. coli BL21/pETDuet-1-glk-zwf harboring the key enzyme genes glk and zwf for efficient NADPH regeneration was successfully constructed. The optimal process condition for the production of NADPH by recombinant strain was obtained via orthogonal experiment， and was as follows: induction temperature 20 ℃， IPTG concentration 0.25 mmol/L， inoculum size 1.5% and loading liquid volume 100 mL/250 mL. The production of NADPH reached 151.79 μmol/L after induction for 48 h in a 1 L shake flask under the optimal condition. Conclusion: This study provides a theoretical basis for efficient NADPH regeneration.