Abstract: The objective calculation of the ecological footprint of water resources in the urban agglomeration in the middle reaches of the Yangtze River was of great significance for assessing the profit and loss of regional water resources and measuring the sustainable use of water resources. In this study, the equivalence factor of the water ecological footprint model was revised according to the “province hectares”. Then, this parameter was applied to the urban agglomeration in the Middle Reaches of the Yangtze River to calculate the water resource ecological footprint and its Spatio-temporal evolution patterns from 2000 to 2019. Finally, based on the Geodetector, the driving factors of the evolution of the ecological footprint of water resources in the urban agglomeration in the middle reaches of the Yangtze River from the four aspects of natural resources, social economy, technological level, and water resources environment was analyzed. The results indicated that:(1) The water resources ecological footprint of the urban agglomeration in the middle reaches of the Yangtze River showed a fluctuating growth trend from 2000 to 2019, with a growth rate of 34.63%. (2) There were significant spatial differences in the ecological footprint of water resources in the urban agglomeration in the middle reaches of the Yangtze River. The water resources ecological footprint in the Wuhan City Group was higher in the north than in the south, and the Poyang Lake City Group showed a distribution trend of “high in the west and low in the east.” The northern and southeastern regions of the water resources ecological footprint in the Chang-Zhu-Tan City Group were higher in the north and southeast than in the central part. (3) The intensity of the ecological footprint of water resources was the main driving factors, and the interaction factors with the highest explanatory power were the intensity of water ecological footprint and GDP. The results could provide a scientific theoretical basis for decision-makers to formulate water resources management policies according to local conditions.