Eight resins were tested systematically to select the best one for the chromatography separation of gamma-aminobutyric acid (GABA) and monosodium glutamate (MSG). Then the influence of the ratio of length to diameter of column, flow rate of mobile phase and column temperature on the separation efficiency of GABA and MSG were investigated in terms of resolution and recovery. Finally, the separation efficiency of fermentation broth was concretely studied. The results showed that the resin QY-HG01 separated GABA and MSG well. The best separation efficiency was achieved under the operating conditions of ratio of length to diameter 25, flow rate of mobile phase 2.5 mL/min and column temperature 50℃ through the single factor experiments. Injected with the pretreated fermentation broth, it was found that GABA and MSG were separated well. The decolorization rate of the fermentation broth was 84.9%, and the desalinization rate was 99.2%. The purity of GABA increased from 24.9% in the broth to 80.4% in the eluent with a recovery yield of 89.3%.