To establish an analytical method for simultaneously determining the contents of 15 amino acids and analyze the content of amino acids in Paris polyphylla var. yunnanensis fibril from different places. The sample was tested by UPLC-PDA instrument, and the amino acids were separated on a ACQUITY UPLC BEH C₁₈ column (1.7 μm，2.1 mm×150 mm) with gradient elution of 0.1 mol/L sodium acetate buffer (adjusted to pH 6.5 with acetic acid)-acetonitrile (97∶3) (A) and acetonitrile-water (4∶1) (B). UV detection wavelength was set at 254 nm and the flow rate was set at 0.2 mL/min. The kinds of 15 amino acids in P. polyphylla var. yunnanensis fibril were determined and showed a good linear relationship within the linear range (r ≥ 0.999 7),and the average recovery RSD was less than 3.00%. The content of aspartic acid and glutamic acid was the highest in all producing areas,and the average content of amino acid in the fibrous roots of cultivated P. polyphylla var. yunnanensis was higher than that of wild. In this study,we established a method to analyze and evaluate the amino acids in P. polyphylla var. yunnanensis fibrous roots,and provided a scientific basis for the development and utilization in P. polyphylla var. yunnanensis fibrous roots resources.