In view of the mutants of lactic acid bacteria obtained by mutagenesis were massive and the traditional determination of GABA content in 96-well plates through spectrophotometer was low efficiency and high cost,microplate reader was used to determine the content of GABA quickly and efficiently herein and the main influencing factors of the method was optimized based on the Berthelot reaction. The optimum condition for the GABA determination was as follows: 0. 75mL 0. 2mol /L pH10. 0 borate buffer,2. 5 mL 6% redistilled phenol,and 2mL NaClO solution with5. 2% available chlorine. Under this condition,the higher concentration of GABA could be detected by microplate reader. The precision test was implemented simultaneously. Finally,in order to verify the feasibility of this method,75 mutant strains were fermented in 96-well plates and then respectively measured by spectrophotometer and microplate reader. The data regression showed a significant relationship between these two methods,and the correlation was0. 9493. The experimental results showed that microplate reader can simply,rapidly and accurately measure the content of GABA. It also laid the foundation for high-throughput selecting and breeding of high-yield GABA from lactic acid bacteria in future.