Freshwater ecosystem services supply and demand are important for achieving Sustainable Development Goal 6 of clean water. Urbanization has significantly impacted the supply, demand, and flow of freshwater ecosystem services due to various human activities. However, the changes in freshwater ecosystem service flow (FESF) under the influence of urbanization were still unclear. This study examines the highly urbanized Dongjiang River as an example and maps the supply-flow-demand network of freshwater services based on social-ecological network analysis. The results indicate that freshwater supply has increased by 0.21 billion m3 per year, while there is a decreasing trend of 0.17 billion m3 per year in demand. The FESF approach reveals that 42%–60% of imbalanced sub-watersheds rely on artificial water diversion projects to alleviate water deficits under dynamic flow conditions. Approximately 15.6% of the region experiences water insecurity, driven by urbanization-induced demand for water diversion. This increased demand puts stress on downstream sub-watershed water supply and elevates the risk of flow reduction in the main stream. The study highlights the need for sustainable water management strategies to ensure freshwater security in large river basins within urbanized areas worldwide and to address the challenges posed by uncertain urban expansion.