Abstract: To obtain lactic acid bacteria with high γ-aminobutyric acid (GABA) production capacity, this study utilized plant-derived strains from the Food Science and Technology College of Hunan Agricultural University as the source. A combined method of thin-layer chromatography and high-performance liquid chromatography was employed to screen for GABA-producing lactic acid bacteria and to optimize cultivation conditions. A lactic acid bacterial strain designated as 729 exhibited high GABA production capacity, achieving a yield of 1.699±0.066 g/L. Morphological observation, physiological and biochemical identification, and 16S rDNA gene sequence analysis confirmed that this strain was Lactobacillus plantarum. Further studies revealed that the strain maintained optimal growth at pH4.0 and 8% NaCl concentration, with survival rates of 81.5%±1.1% in simulated gastric fluid and 88.7%±0.8% in simulated intestinal fluid, indicating a potential for gastrointestinal tolerance. Based on these findings, single-factor, orthogonal, and response surface experiments were conducted to optimize the nutritional and fermentation conditions affecting GABA production capacity of the strain. The results demonstrated that under the following culture medium conditions—fructose 20 g/L; yeast extract and fish meal peptone (1:1, total concentration 15 g/L); L-glutamic acid sodium 25 g/L; and sodium succinate 2 g/L; sodium acetate 5.0g/L; ammonium citrate tribasic 2.0 g/L; dipotassium hydrogen phosphate 2.0 g/L; MgSO₄·7H₂O 0.2 g/L、MnSO₄·4H₂O 0.05 g/L; andTween-80 1.08 g/L-combined with initial pH of 6.5, a cultivation temperature of 34.2 °C, an inoculation rate of 4.1%, and a fermentation duration of 97 h, the GABA yield reached 16.349±0.030 g/L, representing an approximately 8.6-fold increase compared with pre-optimization levels. This study provides a theoretical foundation and microbial strain resources for the development of high-yield GABA-producing lactic acid bacteria and their applications in functional foods.