The gamma amino butyric acid (GABA) is a chemical messenger and is essential for the health of the brain and muscles. Some lactic acid bacteria (LAB) have the potential to function as psychobiotic cultures because they can produce significant amounts of neuroactive compounds like GABA. Psychobiotics are known to alter bidirectional communication between the gastrointestinal tract and the central nervous system. In the present study, the Limosilactobacillus reuteri (L. reuteri) strain, isolated from human breast milk, was used to detect the GABA-producing glutamic acid decarboxylase (gad) gene and GABA production. PCR, HPLC and UHPLCQ-TOF-MS approaches were applied to identify the gad gene, GABA content, and bioactive compounds produced by the bacterial strain, respectively. Additionally, the whole genome was sequenced to better understand the strain's psychobiotic and technological genomic properties. The gadB and gadC genes were confirmed in plasmid 1 of the whole genome. The complete genome sequence of L. reuteri comprises the genome length of 2,087,202 bp with 51.6 percent of G + C content. The results indicate that L. reuteri can be used as a starter culture for the production of GABA-enriched functional foods as well as psychobiotics for health benefits.