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Gamma‐aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. The production GABA from the cheaper glutamic acid is a valuable process. Rice bran is a potential source of glutamic acid decarboxylase which was therefore selected for production of gamma‐aminobutyric acid (GABA) using lactic acid bacteria (Lactobacillus brevis (Lb.brevis) VTCC‐B397). The influences of treatment of defatted rice bran with α‐amylase, Alcalase (Al), Flavourzyme (Fl) and their combinations at different concentration ratios on the GABA synthesis were examined. With α‐amylase impacts, concentration of GABA (6.41 mg mL−1) was about 2.38 times higher than the control sample (2.69 mg mL−1) under hydrolysis conditions of pH 6.5, temperature of 95 °C and α‐amylase concentration of 0.15% (v/w) for 45 min. The α‐amylase hydrolysis as prerequisite for protein hydrolysis using Al: Fl (ratio Al: Fl of 3: 7, concentration of 2% (w/w), pH = 8, temperature of 50 °C) reached the highest GABA synthesis efficiency, at 8.43 mg mL−1. Results showed that the enzyme treatment positively affected the fermentation using Lb. brevis for GABA biosynthesis from defatted rice bran.