An ATP regeneration system between two microorganisms, in which recombinant E. coli II-1 and S. cerevisiae WSH-J702 were used as the biosynthesis enzymes source of glutathione and ATP, respectively, was constructed by using glucose as an energy substrate. Yeast cells after permeable treatment could hardly consume glucose. The glycolysis pathway of yeast was started by adding 1.0 mmol/L AMP and 0.05 mmol/L NADH simultaneously. GSH biosynthesis in the coupling system was improved with increasing glucose concentration to 400 mmol/L, where the concentration of GSH reached 10.4 mmol/L (3.2 g/L). Both the coupling system and the non-coupling system with ATP added could hardly produce GSH under the absence of magnesium. The low efficiency of ATP regeneration system constructed was probably caused by the chelation between magnesium and ATP. By adding magnesium to the coupling system, a final concentration of GSH, 14.3 mmol/L (4.4 g/L), was achieved at 6 h of reaction.