Gut microbiota (GM) alterations have been implicated in autism spectrum disorder (ASD), yet the specific functional architecture remains elusive. Here, employing multi-omics approaches, we investigate stool samples from two distinct cohorts comprising 203 children with mild ASD or typical development. In our screening cohort, regression-based analysis for metabolomic profiling identifies an elevated γ-aminobutyric acid (GABA) to glutamate (Glu) ratio as a metabolic signature of ASD, independent of age and gender. In the validating cohort, we affirm the GABA/Glu ratio as an ASD diagnostic indicator after adjusting for geography, age, gender, and specific food-consuming frequency. Integrated analysis of metabolomics, 16S rRNA sequencing, and metagenomics reveals a correlation between overrepresented Escherichia and disrupted GABA metabolism. Furthermore, we observe social behavioral impairments in weaning mice transplanted with E. coli, suggesting a potential link to ASD symptomatology. Collectively, these findings provide insights into potential diagnostic and therapeutic strategies aimed at evaluating and restoring gut microbial neurotransmitter homeostasis.