Gamma-aminobutyric acid (GABA) have attracted growing interest in recent decades due to its multiple health benefits with numerous and important physiological functions, such as anti-cancer, anti-hypertension, anti-diabetes, antioxidant and anti-inflammation. GABA is produced by lactic acid bacteria, which can produce a high amount of this amino acid. The food industry has leveraged this capacity to develop functional foods enriched with GABA. For this reason, the current study reports the isolation of lactic acid bacteria (LAB) and their screening for GABA production from monosodium glutamate (MSG) using pre-stained thin-layer chromatography (TLC). The isolate MGOLIV.25 was selected as a high GABA producing bacterial strain. The GABA content of MGOLIV.25 was detected by Gas chromatography–mass spectrometry (GC-MS) to be 9.02 mg/ml. The isolate MGOLIV.25 was identified biochemically and molecularly as Lacticaseibacillus casei. The findings of this study suggest that the isolate Lacticaseibacillus casei could be used for developing of healthy food products rich with GABA.