It is important to create a win-win situation on both economic development and ecological protection. For better ecological management and protection in the future, it is essential to identify the trade-offs and synergies of multiple ecosystem services (ESs) at differently spatial scales. The relationships of ESs varied over time and spatial scales, it is important to study the spatio-temporal dynamics of relationships to realize the trends of ESs change and determine appropriate scale to manage. However, the spatio-temporal dynamics of ESs relationships in the Chinese large basin remain a challenge, as information on adaptable unit to ESs management is often insufficient. Therefore, the purpose of this research is to identify relationships of ESs on sub-watershed and county scales. Six key ESs in Nansihu Basin were selected to simulate and evaluate by InVEST (Integrated Valuation of Ecosystem Services and Tradeoffs) model since the reform and opening-up (1980-2018), which included water yield (WY), water purification (WP), sediment delivery ratio (SDR), carbon storage (CS), habitat quality (HQ), and crop pollination (CP). In this paper, the spatial distributions of six ESs were analyzed and the hotspots of each ES were identified by hot spot analysis in ArcGIS 10.2. Moreover, the spatial and temporal dynamics of relationships among six ESs were analyzed through correlation and principal component analyses via the correlation and principal component analyses. And further analyses were performed on different scales to the relations. The results displayed as follow:(1) in the Nansi Lake Basin, the WY was increasing from 1980 to 2018, while WP, SDR, CS, HQ and CP were decreasing during this periods. (2) The land use types in eastern basin were mainly forest and grassland, which possessed high levels of CS, HQ and CP. The areas with construction land suggested high levels of WY and SDR. (3) There was a significant trade-off relationship between WY and WP, as well as strongly synergetic relationships between WP and HQ, CS, CP occurred in the basin. (4) There was a great difference in relationships between sub-watershed and county scale. Trade-offs mainly occurred between the provisioning service (such as WY) vs the regulating services (such as WP and SDR) and provisioning service (such as WY) vs the supporting service (such as HQ) on sub-watershed scale. While on the county scale, there was a synergetic relationship between the provisioning service and regulating services vs supporting services (such as WY, WP, CS, HQ, CP). Our research provided an assessment framework to spatio-temporal dynamics of ESs relationships, which suggested that sub-watershed unit was suitable for ecological management. Based on the results of different spatial scales, this research provided scientific support for managers to formulate corresponding strategies at appropriate spatial scale and promote eco-economic coordinated development in the Nansihu Lake Basin.