Amino acids are indispensable components of living organisms. The high amino acid content in Nitraria tangutorum Bobr. fruit distinguishes it from other berry plants and is of great significance to its nutritional value. Herein, using 10-ethyl-acridine-3-sulfonyl chloride as a fluorescent pre-column labeling reagent, a method for the efficient and rapid determination of amino acid content in N. tangutorum by pre-column fluorescence derivatization and on-line mass spectrometry was established and further validated. The limits of detection (signal-to-noise ratio = 3) were between 0.13 and 1.13 nmol/L, with a linear coefficient greater than 0.997 and a relative standard deviation between 1.37% and 2.64%. In addition, the method required a short analysis time, separating 19 amino acids within 20 min. Subsequently, the method was used to analyze the amino acid content of Nitraria tangutorum Bobr. from tissues retrieved from seven regions of the Qinghai-Tibet Plateau. Nitraria tangutorum Bobr. was shown to contain a large amount of amino acids, with the total content and main amino acid varying between the different tissues. This research supports the nutritional evaluation, quality control, and development and utilization of Nitraria tangutorum Bobr.