Abstract: Ecological networks can be optimized to mitigate landscape fragmentation for better connectivity in the sustainable development of modern agriculture. Different natural resources and human activities can also be considered in various regions. Taking the Weihe River Basin as the study area, this study aims to optimize ecological networks using supply-demand ecosystem service and topological structure analysis. Firstly, the ecosystem service supply and demand ratio (ESDR) was quantitatively measured for five ecosystem services in 2000, 2010, and 2020 using the InVEST model and the ecological process equation. Secondly, the landscape ecology and complex network were introduced to construct the spatial and temporal patterns of the ecological network and the topological structure using morphological spatial pattern analysis (MSPA), connectivity analysis, circuit theory model, and connectivity robustness formula. Thirdly, the ecological network was optimized to rank and screen the ecological pinch points and barriers in the ecological corridor, according to the correlation between the ESDR and the topological comprehensive importance of the nodes at the ecological patches. Finally, the optimization of the ecological network was evaluated on the connectivity robustness The results show that: 1) The average ESDR of food production, soil conservation, and water yield in 2020 increased by 70%, 7%, and 215%, respectively, compared with 2000. While the carbon storage and habitat quality decreased by 97% and 1%, respectively. There was the most significant increase in the supply or decrease in the demand for water yield services. 2) The number and ratio of ecological patches to the total area were 125 and 36%, respectively. The ecological resistance exhibited the spatial patterns of “low in the south and high in the north”, indicating the decreasing trend of resistance in the north. Furthermore, there were approximately 280 ecological corridors with an average length of 15 km. The ecological quality of the basin was improved significantly. However, there was no variation in the number of the ecological network elements. 3) The average values were 0.160, 0.168, and 0.150, respectively, in the structural connectivity robustness of the ecological network in 2000, 2010, and 2020, indicating better stability in the ecological network in 2010. The topological comprehensive importance of ecological patches was positively correlated with the ESDR in each type of service. The highest correlation was with the ESDR of carbon storage at 0.51. 4) The optimized ecological network had high connectivity robustness in the face of deliberate attacks. Additionally, there was a more significant “emergence” of the optimized network within a certain range of attacking nodes, indicating the stronger resilience and buffering capacity. The spatial and temporal patterns of the ecological network can be expected to facilitate decision-making and strategic planning. The finding can provide a scientific foundation to optimize the ecological network structure for the resistance to the ecosystem in the WRB. Effective support can also be offered for the ecological spatial planning of the basin.