Exploring the constraint relationship between physical geographic features and urbanization on ecosystem services is important for managing and optimizing regional ecosystem services. Taking Anhui Province as an example, we assessed the spatial and temporal evolution characteristics of five types of ecosystem services (habitat support, water production services, soil conservation, NPP, and carbon fixation) and five types of urbanization levels (population, economic, social, ecological, and spatial) in 2000, 2010, and 2020, and integrated the constraint line method, bivariate spatial autocorrelation model, and spatial regression model to measure the relationship between ecosystem services. The spatial constraints between ecosystem services and urbanization level and natural topography in Anhui Province were measured using the constraint line method, bivariate spatial autocorrelation model and the spatial regression model. The results show that: (1) the spatial distribution of the five types of ecosystem services in Anhui Province is characterized as “low in the north and high in the south”. At the provincial level, the five ecosystem services in southern and central Anhui Province are synergistic, while the five ecosystem services in northern Anhui Province show a trade-off; (2) topography has different effects on the five ecosystem services with “exponential” effects on water production services and NPP, “positive convex” effects on habitat support, and “positive convex” effects on habitat support”; (3) the bivariate global autocorrelation Moran’s I index between ecosystem services and urbanization level in Anhui Province is significant, confirming that ecosystem services and urbanization are spatially related, where the development of population urbanization, spatial urbanization, economic urbanization, and social urbanization leads to the decrease in ecosystem services, and ecological urbanization promotes the increase in ecosystem services. In the spatial regression model, the Spatial Lag Model passed the significance test, indicating that there is a spatial spillover effect between ecosystem services and urbanization. That is, changes in ecosystem services are influenced not only by their own urbanization elements, but also by urbanization elements in neighboring units or more distant units. Exploring the constraints of ecosystem services and identifying their interaction with urbanization can provide a scientific basis for land-use optimization, adjusting management measures and achieving regional sustainability.