Rapid urbanization in urban agglomeration areas puts tremendous pressure on the eco-environment, and balancing urbanization development and ecological quality protection is crucial for regional high-quality development. However, current research lacks a multi-scale assessment of ecological quality and urbanization in urban agglomeration areas. Here, we assessed the multi-scale ecological quality and urbanization level of the Middle Reaches of the Yangtze River Urban Agglomerations (MRYRUA) from 2001 to 2021 based on multi-source remote sensing imagery by constructing the remote sensing-based ecological index (RSEI) and the comprehensive nighttime light index (CNLI), respectively, and analyzed the coupling coordination degree (CCD) of the two using the CCD model. The following conclusions were obtained: (1) There are spatial differences in the ecological quality of pixel, county, and municipal scales. Multi-dimensional analysis indicates that the RSEI shows an increasing trend, and the ecological quality of the MRYRUA has generally improved in the past 20 years. (2) The DN values of nighttime light and the three light indices have increased to varying degrees at different scales, among which the average value of CNLI has increased from 0.02 in 2000 to 0.12 in 2021. The urbanization level of the MRYRUA is constantly improving, but there is an obvious development imbalance. (3) The ecological quality and urbanization level developed in a more coordinated direction at different scales, but the urbanization level lagged behind ecological quality as a whole, and the two were negatively correlated in space. The results of this study can guide the formulation of ecological protection and high-quality development policies in the MRYRUA.