Glutathione (GSH) is an important reducing agent in the living cells. It is synthesized by a two-step reaction and requires two molecules of adenosine triphosphate (ATP) for one molecule GSH. The enzymatic cascade reaction in vitro is a promising approach to achieve a high titer and limit side reactions; although, a cost-effective phosphate donor for ATP regeneration is required. Triphosphate (PolyP((3))), tetraphosphate (PolyP((4))), and hexametaphosphate (PolyP((6))) were investigated in this study. Triphosphate inhibited the bifunctional GSH synthetase (GshF) from Streptococcus agalactiae, while no significant inhibition was observed by adding hexametaphosphate. The polyphosphate kinase from Corynebacterium glutamicum was hence investigated to use hexametaphosphate for regeneration of ATP. Further, the orthogonal experiment, which includes seven factors (buffer concentration, pH value, ADP concentration, GshF dosage, polyphosphate kinase (PPK) dosage, reaction temperature, substrate ratio of amino acid, and reaction times), indicated that the capacity of buffer is the most significant factor of the reaction conditions for enzymatic production of glutathione coupling with a PPK-based ATP regeneration system. After optimizing the Mg2+ concentration, the reaction was scaled up to 250 mL in a stirred reactor with pH feedback control to stabilize the pH value of reaction system and nitrogen protection to avoid the oxidation of product. A yield of 12.32 g/L was achieved. This work provided a potential GshF-based enzymatic way coupling the PPK-based ATP regeneration to product GSH in the optimal conditions towards cost-effectiveness at the industrial scale.