Abstract: In order to improve the yield of γ-aminobutyric acid (GABA), the co-fermentation conditions of Enterococcus faecium AB157 and Saccharomyces cerevisiae SC-125 were optimized by one-factor-at-a-time method and response surface methodology (RSM). Simultaneously, the enzyme activity of glutamate decarboxylase (GAD) was analyzed under optimal conditions in co-fermentation and single strain fermentation systems, and the mechanism of high GABA yield was explored by adding cell-free supernatant (CFS). The optimization results showed that when the overall quantity of inoculum was 2% (V/V), the optimal co-fermentation conditions were as follows: The fermentation temperature was 35 ℃, the inoculum proportions of E. faecium AB157 and S. cerevisiae SC-125 was 5:1 (V/V), and the L-monosodium glutamate concentration was 12 g/L with shaking fermentation for 96 h. In addition, the yield of 6.55 g/L GABA was 1.78 times higher than in single strain fermentation systems. The GAD enzyme activity analysis showed that co-fermentation could significantly improve GAD enzyme activity. Meanwhile, GABA yield could be significantly increased by adding CFS of E. faecium AB157 or S. cerevisiae SC-125. This study served as a theoretical foundation for the discussion of the co-fermentation of E. faecium and S. cerevisiae to increase GABA yield and the mechanism of high GABA yield.