Abstract: The Hanjiang River Basin is not only the water source of China's South-to-North Water Transfer Project, but also an important ecological function area of the country. The interaction of ecosystem service in Hanjiang River Basin plays an important role in coordinating regional development and environmental protection. In this paper, we mapped the ecosystem service of soil conservation, carbon storage and food supply in Hanjiang River Basin from 2000 to 2015 by RS and GIS technique, and then studied the trade-off and synergy between the three ecosystem services based on the spatial sampling method. The results are shown as follows: ① In 2000-2015, the soil conservation in Hanjiang River Basin decreased in fluctuations. The high value regions of soil conservation are located in the upper reaches in Hanjiang River Basin, where is forest and grassland cross-area. The low value is in the single-type areas such as forest land or cultivated land in the middle-lower reaches of Hanjiang River Basin. In addition, the annual variation of carbon storage is small, the distribution characteristics of carbon storage are consistent with that of soil conservation. Food supply is growing rapidly, with high region located in plains of the middle-lower reaches, and low value regions in the mountainous and basin areas of upper reaches. ② When analyzing trade-offs and synergies, there are trade-off relationships between the ecosystem services of carbon storage and food supply, soil conservation and food supply. In contrast, the interaction between carbon storage and soil conservation is prone to synergistic. It indicates that trade-off and synergy between multiple ecosystem services have spatial heterogeneity. ③ The results of spatial hotspot mapping show that, from 2000 to 2015, the proportion of category 0 hotspot present a decreasing tendency, and grassland is the main land type of 0 class; The category 1 hotspot distribates in the cultivated land, account for the largest proportion and present an increasing trend, which dominant ecosystem service is food supply. The category 2 hotspot decreases for some extend, mainly distribute in forest areas of the upper-middle reaches in Hanjiang River Basin. The ecosystem services of the category 2 are carbon storage and soil conservation. Moreover, the proportion of category 3 hotspot is very small. These findings contribute to revealing regional difference of ecosystem service.