Objective：To establish a simple，accurate and reliable method for the batching detection of amino acid neurotransmitters and taurine in brain. Methods：The amino acids were detected by using A300 amino acid analyzer. The protein was precipitated by using 10% sulfosalicylic acid. Lithium salt reagent was used as mobile phase and ninhydrin was used as color reagent. The detection wavelength was 570 nm. The resolution，linear range，detection limit and quantitative limit，intraday precision and recovery，daytime precision of the method were determined. SPF-class KM mice at the same age were selected and divided into two groups：male and female. Three in each group were decapitated and executed，and each mouse was separated the cortex and hippocampus from the brain on ice. The free amino acid samples of mouse brain tissue were prepared by homogenization，centrifugation，protein precipitation，dilution and membrane transmembrane. The contents of aspartic acid（Asp），glutamic acid（Glu），glycine （Gly），gamma-aminobutyric acid（GABA）and Tau in cortex and hippocampus were determined by amino acid analyzer. Results：Five target amino acids in standard samples，cortical and hippocampal samples of male and female mice were well separated. Within the concentration range of 8~400 μmol·L-1 ，the instrument had a good linear relationship（R2 ≥0.999）. Detection limit was 1.29~1.86 μmol·L-1 . Quantitative limit was 4.29~6.20 μmol·L-1 . The intraday relative standard deviation（RSD）of standard samples were less than 2.23%，the intraday RSD of brain tissue samples were less than 2.76% and the recovery of the spiked samples were in a range of 91.52%~102.51%. 5 kinds of amino acid content in the sample were accurately obtained by plotting the peak area curve of the standard sample. No statistically significant difference was found between the male and female mice（P>0.05）. Conclusion：The method is simple，accurate， reliable and suitable for batching detection of free amino acid content in mouse cortex and hippocampus.