Anatomical, histochemical and phytochemical methods were used to investigate the structure, localization and dynamic changes of total saponin and senegenin of vegetative organs in Polygala tenuifolia Willd. Histochemical localization results showed that saponin accumulated mainly in parenchyma cells of vegetative organs. The phytochemical results also showed that the saponin accumulated in the vegetative organs of P. tenuifolia, with higher content in roots and lower content in the aerial parts that included stems and leaves. The saponin content and dry weight of the vegetative organs of P. tenuifolia had dynamic variance at the developmental stages and all reached the highest level in the post-fruit period. Hence, the roots and aerial parts should be gathered in August to make full use of the plant. As the root is the main medicinal organ of P. tenuifolia, the content of total saponin and senegenin of different aged and different parts of the root were determined. The content of total saponin and senegenin exhibited a sustained decreasing trend with increasing root age; therefore, the annual roots had high quality. The content of total saponin and senegenin in different parts of the root showed obvious variation. The content in the "skin areas" was much higher than that of xylem. The results offer a theoretical basis for determining the appropriate harvesting stage and a reasonable harvest of P. tenuifolia.