Response surface methodology (RSM) was employed to study the effects of four medium components (L-glutamic acid, citric acid, glycerol and NH4Cl) on the production of poly-γ-glutamic acid (PGA) in flask cultures by Bacillus licheniformis CCRC 12826 (ATCC 9945) with initial medium pH 6.5. The optimal PGA yield (35.34 g/L, average of four repeats) was appeared at the region where the respective concentrations (in g/L) of glutamic acid, citric acid, glycerol and NH4Cl were around 34.0, 26.0, 146.0 and 11.0, respectively. The optimized composition derived from RSM regression was (in g/L) glutamic acid 34.70, citric acid 27.00, glycerol, 145.45 and NH4Cl 10.49. With this composition, the PGA production was 33.20 g/L (average of eight repeats) after 96 h of cultivation, while the predicted maximum production was 35.52 g/L. The optimized medium resulted in significant increase of the PGA yield by B. licheniformis CCRC 12826 in shake-flask cultivation without any feeding process. In comparison with glutamic acid, citric acid and glycerol, NH4Cl represents a crucial role in PGA biosynthesis.