To study the accumulation law of pharmacodynamic substances,12 batches of Polygalae Radix from different producing areas and different growth years were collected,and the contents of water and extract were determined. The contents of polygalaxanthone Ⅲ (PX Ⅲ),3,6′-disinapoylsucrose (DISS),and tenuifolin (TEN) were determined by HPLC,and the fingerprint was established. The results showed that the root length and diameter of Polygalae Radix increased with the increase of the growth year,and the increasing trend of diameter was more significant. There are obvious differences in the water content of Polygalae Radix from different producing areas. The water content of Polygalae Radix from Fenyang is about 4.0%,that of Xinjiang is about 8.2%,which is higher than that from Fenyang,and that of 12 batches of Polygalae Radix is less than 12%. The content of extract is between 38%-48%. The content of PX Ⅲ was 0.11%-0.18%,and the content of DISS was 0.50%-0.94%,The content of TEN ranged from 1.37%-2.40%. The accumulation law of PX Ⅲ and DISS was similar. The content of TEN in the second and fourth years was close;The total content of index components of Polygalae Radix produced in Xinjiang accumulated significantly,and the maximum value appeared in 3 years of growth. The fingerprint similarity of 12 batches of samples ranged from 0.823-0.962,with a total of 35 common peaks,and 2 of them were identified. There was no significant difference between the fingerprint similarity of Fenyang and Xinjiang,and the similarity of spring harvest was higher than that of the autumn harvest. In brief,the results of this study provide a reference for the cultivation and harvest time of Polygala tenuifolia. The content determination of three active components combined with fingerprint could provide a reference for the quality control and evaluation of Polygalae Radix.