Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is widely known to have multiple physiological functions. The present study aimed to investigate the cultivation parameters for GABA production by a lactic acid bacteria (LAB) strain isolated from a tuna gut sample. Among 60 tuna gut LAB, only 7 Limosilactobacillus fermentum isolates, i.e. NG01, NG12, NG13, NG14, NG16, NG23, and NG27, were capable of GABA fermentation, with NG16 being the most potent GABA producer. The GABA production by isolate NG16 was therefore thoroughly characterised. The optimal batch culture conditions for GABA production were an initial cell density of 5×106 CFU mL−1, a monosodium glutamate concentration of 2%, an initial pH of 7, a fermentation temperature of 35 °C, and an incubation time of 96 h. Under this cultivation conditions, NG16 produced a maximum GABA yield of 25.52 ± 0.41 mM.