To study and establish the fingerprint of Polygala tenuifolia by RP-HPLC.The fingerprint of P. tenuifolia was built by using Kromasil C18 as column and acetonitrile-0.05% phosphoric acid aqueous in gradient as mobile phase. The flow rate was 1.0 mL x min(-1), and the detecting wavelength was set at 318 nm. Total 14 batches of P. tenuifolia from different habitats were detected, and different commodities and preparative methods were compared.Samples from different habitats were of high similarities, and the qualities of "yuanzhitong" and "yuanzhirou" were better than that of "yuanzhigun", while the fingerprints of P. tenuifolia with different preparative methods have not obvious differences. Moreover, the standard fingerprint of P. tenuifolia was originated from the "Computer Aided Similarity Evaluation" software, and 29 common peaks existed in the fingerprint. Each peak in the fingerprint was separated very well under the above chromatographic condition, with good accuracy, stability and repeatability, satisfied to the requests of fingerprint.The RP-HPLC fingerprint method can be used in the quality control of P. tenuifolia.