Optimum production of L-tryptophan by Escherichia coli depends on pH. Here, we established conditions for optimizing the production of L-tryptophan. The optimum pH range was 6.5-7.2, and pH was controlled using a three-stage strategy [pH 6.5 (0-12 h), pH 6.8 (12-24 h), and pH 7.2 (24-38 h)]. Specifically, ammonium hydroxide was used to adjust pH during the initial 24 h, and potassium hydroxide and ammonium hydroxide (1:2, v/v) were used to adjust pH during 24-38 h. Under these conditions, NH4 (+) and K(+) concentrations were kept below the threshold for inhibiting L-tryptophan production. Optimization was also accomplished using ratios (v/v) of glucose to alkali solutions equal to 4:1 (5-24 h) and 6:1 (24-38 h). The concentration of glucose and the pH were controlled by adjusting the pH automatically. Applying a pH-feedback feeding method, the steady-state concentration of glucose was maintained at approximately 0.2 ± 0.02 g/l, and acetic acid accumulated to a concentration of 1.15 ± 0.03 g/l, and the plasmid stability was 98 ± 0.5 %. The final, optimized concentration of L-tryptophan was 43.65 ± 0.29 g/l from 52.43 ± 0.38 g/l dry cell weight.