We have developed an economical and simple cell-free protein synthesis system that produces milligram quantities of proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged protein synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, protein synthesis continued for 6 h and the amount of the accumulated protein reached 1.8 mg/mL.