Lactic acid bacteria were isolated from fish and evaluated for their γ-aminobutyric acid (GABA)-producing abilities. Out of thirty-two isolates, Lactobacillus brevis RK03 showed the highest GABA production ability. The effects of various fermentation parameters including initial glutamic acid level, culture temperature, initial pH, and incubation time on GABA production were investigated via a singleparameter optimization strategy. For industrial large-scale production, a low-cost GABA producing medium (GM) broth was developed for fermentation with L. brevis RK03. We found that an optimized GM broth recipe of 1% glucose; 2.5% yeast extract; 2 ppm each of CaCO3, MnSO4, and Tween 80; and 10 μM pyridoxal phosphate (PLP) resulted in a maximum GABA yield of 62,523 mg/L after 88 h following the addition of 650 mM monosodium glutamate (MSG), for a conversion rate of 93.28%. Our data provide a practical approach for the highly efficient and economic production of GABA. In addition, L. brevis RK03 is highly resistant to gastric acid and bovine bile salt. Thus, the discovery of Lactobacillus strains with the ability to synthesize GABA may offer new opportunities in the design of improved health-promoting functional foods.