[Objective] This study is aimed to scientifically construct the ecological network of urban agglomerations in the middle reaches of the Yangtze River, and to provide scientific basis and theoretical reference for cross-regional ecological protection and collaborative governance. [Methods] Multi-temporal ecological network was conducted based on the framework of "source identification-resistance surface construction-corridor extraction", and network evaluation was done based on network attributes and human activities. [Results] (1) Ecological sources were mainly concentrated in the mountainous of northwestern Hubei Province, junction of Jiangxi and Hunan, and the basin of Poyang Lake basin, with the area reducing from 2.67×10⁷ km² in 2000 to 2.29×10⁷ km² in 2020. The number of ecological corridors decreased from 69 to 42, and the total length decreased from 1.53×10⁴ km to 1.16×10⁴ km. The resistance of study area increased gradually, the center of gravity of network had shifted from Hubei Province to Hunan Province, transforming from a strip distribution of "three horizontals and two verticals" to a concentrated group distribution. The overall degree of aggregation and network connectivity of the ecological network were weakening, and showing a trend of structural contraction and functional weakening overall. (2) In network optimization, 102 ecological "breaking points" were identified, and 17 stepping stones were set based on the distance between the source and destination. A "core protection belt" and an "ecological control belt" were set up within 5 and 15 km of the network, with length of 1 505 km. [Conclusion] In synergistic governance, at the provincial level, Hubei and Jiangxi provinces should improve the cross-provincial ecological compensation mechanism for river basins, while Hunan and Jiangxi provinces should strengthen the judicial guarantee in the implementation of the synergistic mechanism. At the city level, differentiated management and control would be implemented for key management areas, and potential ecological corridors would be planned to promote synergistic governance at the city level. The integration of the optimized ecological network was improved from 0.22 to 0.30, effectively improving ecological connectivity between regions.