Abstract: Objective: To establish a quantitative analysis of multi-components by single-marker (QAMS) with using glutamic acid as the internal reference, the content of 17 amino acids in Morchella esculenta was determined simultaneously. Methods: High performance liquid chromatography (HPLC) was used with acetonitrile-ammonium acetate aqueous solution as mobile phase, gradient elution and glutamic as the internal reference substance was used to establish its relative correction factors (RCF) of other 16 amino acids. The content of 17 amino acids in Morchella esculenta was calculated by using the relative correction factor. Internal standard method was used to determine 17 amino acids in Morchella esculenta. The validity of the QAMS method was evaluated by comparison of the quantitative results of both methods and to validate the durability of this method. Results: The 17 amino acids had a great linear relationship in the linear range, and the correlation coefficients higher than 0.9990, the average recovery rate of 17 amino acids were in the range of 92.42%~101.16%, and the relative standard deviations (RSD) were in the range of 0.25%~1.94%, and reproducibility was good in different experimental conditions (RSD<2.0%); through the comparative analysis of determination results of QAMS and the internal standanrd method, the two methods had no significant difference (P>0.05). The RCF and the relative retention time RSD in different chromatographic columns and chromatographic systems all less than 2.0%, and the durability was good. Conclusion: This method is simple, easy to operate, accurate and can save the cost of content determination. It can be used as a new model for the determination of amino acid content in Morchella esculenta, and can also provide a valuable reference for the quality evaluation of Morchella esculenta.