In this study, we developed a method of liquid chromatography with fluoresce detector (LC-FLD) and on-line mass spectrometry identification (MSI) using 2-[2-(7H-dibenzo [a, g] carbazol-7-yl)-ethoxy] ethyl chloroformate (DBCEC-Cl) as pre-column derivatisation reagent for determination of the amino acids (AAs) in Potentilla anserina L. Separation of the derivatised AA exhibited a good baseline resolution in combination with a gradient elution. All AA derivatives give excellent linear responses with correlation coefficients of >0.9992. The detection limits of each AA were 2.60–24.3 fmol. Eighteen AAs, involving seven essential AAs, were detected in Potentilla anserina L. Quantitative recoveries of the AAs from the Potentilla anserina L. were 84–107%, and the relative standard deviation values were <1.45%. The established method in this study was sensitive and precision enough to separate and quantify AA composition of Potentilla anserina L. Good compositional data were obtained from the analysis of the AAs obtained from Potentilla anserina L. root.