In order to clarify the characteristics of soil priority path and the influence of root on the formation of soil priority path under the cultivation of Pu'er tea in southern Yunnan, we took the typical Pu'er tea producing areas as the research object and shrub grassland as the control, and the relationship between root characteristics of Pu'er tea tree and soil priority path was quantitatively analyzed based on the staining tracer method and image processing technology, and the influence of root on the formation of soil priority path was explained. The results showed that: (1) With the increasing of soil depth, the width of dyeing path in tea field and shrub grassland decreased in different extent. The connectivity of priority path in tea field was worse than that in shrub grassland, and the development degree of priority path in tea field was less than that in shrub grassland. (2) The root mainly distributed in the surface of soil, with the increasing of soil depth, root weight density and root length density of different diameter classes all decreased, and the root length density of diameter class ≤1 mm contributed the most, reaching 84.08%. (3) The width of staining path was significantly correlated with root weight density, root length density of diameter class ≤1 mm and 1~3 mm (P<0.05). The width of staining path of shrub grassland was significantly correlated with root length density of diameter class ≤1 mm (P<0.01), and the correlation coefficient was 0.986. (4) The staining path width had a better fitting effect with the root length density of diameter class ≤1 mm, and the R² was as high as 0.970 6. The fine roots with diameter class ≤1 mm had a greater influence on the formation of priority path. Root is one of the important factors that affect soil priority path. The research on the influence of Pu'er tea tree root on soil priority path in southern Yunnan can provide some scientific basis for soil erosion control and water resources management.