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The analysis of stable nitrogen isotopic composition (δ15 N) of individual amino acid was recognized as an effective method for estimating the trophic level of organisms and detecting the nitrogen flow in food webs. In this study, we evaluated a two-stage procedure of esterification followed by acylation, in which biological samples underwent hydrolysis in acid and the released individual amino acids were derivative into the corresponding N-pivaloyl-iso-propyl ( NPP ) esters for gas chromatography-combustion-isotope ratio mass spectrometric ( GC-C-IRMS) analysis. A total of 13 kinds of individual amino acid derivatives were baseline separated on a nonpolar gas chromatography column (DB-5ms). The amount of sample for each test was not less than 20 ng N on column. High correlations were observed between the δ15 N values respectively obtained by GC-C-IRMS and element analysis-isotope ration mass spectrometry (EA-IRMS). Furthermore, the mean precision of this method was better than 1‰. Cation-exchange chromatograph was used to purify the samples, and the difference of the detection δ15 N values before and after purification by the resin was within 1‰. This method was applied to estimate the trophic level of various natural freshwater organisms from Aha Lake. The present study provided a new idea for the application of stable nitrogen isotope (δ15 N ) in the trophic level estimation of organisms and metabolism analysis of amino acid.