Abstract: Objectives: It is of great significance to explore the ecological effect of urban network process for regional sustainable development. Existing studies have studied the impact of urban network on ecosystem function from the perspective of externality, but paid little attention to the impact of ecosystem network structure. Methods: This paper constructs urban network and ecological network respectively based on night light data and MSPA model. Taking urban agglomeration in the middle reaches of the Yangtze River as the research area, QAP analysis and spatial analysis are used to explore the impact of urban network on regional ecosystem. Results: The conclusions are as follows: 1) From 2000 to 2020, the network spatial growth trend of urban agglomerations in the middle reaches of the Yangtze River has become increasingly significant. The former single-center agglomeration development has shifted to a multi-center and decentralized growth pattern, and the connections between cities have become increasingly close. 2) During the same period, the regional ecosystem network structure became increasingly complex, the number of ecological sources and corridors have increased, and the overall network connectivity efficiency has improved. 3) The growth of urban network has a positive externality on regional ecosystem. From the perspective of structure, the ecosystem structure is affected by the positive effect of urban network development, and the overall network structure has been improved. However, with the continuous advancement of the network development, ecological sources and corridors will gradually be occupied and disturbed, and the flow of matter and energy in the network may be hindered. From the functional point of view, urban network development alleviates the impact of regional ecosystem function. There was a significant negative correlation between ecosystem function and network interaction intensity. Conclusions: The results can provide reference for the coordinated development of urban network process and ecosystem.