A rapid and accurate quantitative method of high performance liquid chromatography (HPLC) with fluorescence detector has been developed for the analysis of 18 kinds of amino acids in fresh tea leaves. The samples were minced and mixed, and extracted with ultra pure water at 90℃ for 20 min. The 6-aminoquinolylN-hydroxy-succinimidyl carbamate (AQC) was used as pre-column derivatization reagent. Gradient HPLC separation was performed on a C18 column (Symmetry C18, 3.9 mm × 15 cm, 4 μm). Good linearity between concentrations and peak areas was achieved in the concentration range of 5.0-250 μmol/L for 18 kinds of amino acids. The method was validated by the analysis of five replicates. The 18 kinds of amino acid standards were spiked in fresh tea leaf samples and the average recoveries were 86.25%-109.05% with relative standard deviations (n = 5) ranging from 6.03% to 10.56%. The limit of detection (LOD) for the analytes was 0.05-1.27 μmol/L. The method was successfully applied to the analysis of the 18 kinds of amino acids in fresh tea leaves from east Dongting and west Dongting mountains in Suzhou. The results indicate that the method is simple, rapid, precise and reliable.