The research on the interaction between ecosystem services and urbanization is a hot and frontier field of international human-earth system research. This study selected 325 counties in the middle reaches of the Yangtze River, and estimated the ecosystem services balance and the level of urbanization in 1995, 2005, and 2015 through the ecosystem services supply and demand balance assessment matrix method and urbanization-related indicators. Meanwhile, the bivariate spatial autocorrelation model and spatial measurement model were used to explore the spatial relationship between ecosystem services balance and urbanization. The results showed that:(1) during the study period, the ecosystem services balance in the middle reaches of the Yangtze River has deteriorated, while the level of urbanization continued to rise. (2) There was a significantly negative correlation between the ecosystem services balance and urbanization, and the spatial clustering patterns of different regions showed heterogeneity. (3) The spatial measurement model with added control variables was proved to be more suitable for explaining the spatial relationship between the ecosystem services balance and urbanization. It showed that the impact of urbanization on the ecosystem services balance had a negatively external effect, and this negatively external effect gradually weakend when urbanization reached a certain stage. Finally, we suggest that in the process of urbanization, ecological space should be managed as a whole; cross-regional cooperation needs to be tried; and ecological and economic resources should be shared and complemented.