Abstract: The spatio-temporal characteristics of ecosystem services in Dongting Lake Basin during 2000-2020 were investigated by InVEST model and GIS spatial analysis at sub-watershed scales. The trade-off/synergy analysis of three key ecosystem services such as water yield, water purification and carbon storage was carried out using the coupling coordination degree model and the geodetector model. The results showed that: (1) Water yield services and water purification services were continuously strengthened, while carbon storage services were continuously weakened. In terms of spatial variability, water yield in the southeast was higher than that in the northwest in 2000 and 2010. In 2020, water yield in the northwest was the maximum. The distribution of N and P output was less in the upstream than in the downstream, and higher in the east than in the west. The carbon storage was the least in Dongting Lake area and the highest value was concentrated in the upstream area. (2) From 2000 to 2020, the pairwise ecosystem services in the whole basin were at a moderate level of synergy, and developed to a good level of synergy. There was an obvious difference in the spatial and temporal distribution pattern of pairwise ecosystem services. (3) The formation of nonlinear dynamic relationship between ecosystem services in Dongting Lake Basin was influenced by natural and human factors, including terrain and topography, climate change, land use type and distribution pattern, and human interference. The degree of influence of natural factors was greater than human factors. These results may provide theoretical basis for the construction of ecological protection compensation mechanism in Dongting Lake Basin.