Analysis of γ-aminobutyric acid (GABA) in fermented milk by HPLC was optimized. GABA was extracted from fermented milk with trichloroacetic acid - water solution. Sample solution was heated at 60 ℃ for 2 h, and centrifugated at 10000 r/min for 10 min. The most efficient and rapid detection method of GABA in fermented milk was established by adjusting the elution mode of mobile phase, mobile phase ratio, pH, and column temperature, as well as using amino acid column, automatic injector and instrument automatically derived procedures. The limit of detection (LOD) (S/N=3) and limit of quantification (LOQ) (S/N=10) for GABA were 0.005 μg/mL and 0.02 μg/mL, respectively. The linear range was 0.0025~500 μg/mL with correlation coefficient (R2) of 1. This method could provide shorter detection time, more accurate results, less reagent waste and wider applicability. Meanwhile the results are satisfying for analysis of GABA in functional food samples, red yeast rice samples, and lactic acid fermentation broth samples.