Climate change and human activities combine to alter river hydrology, thereby threatening the health of river ecosystems. Quantifying the impacts of climate change and human activities on ecological flow assurance is essential for water resource management and river ecological protection. However, fewer studies quantify the impacts of climate change and human activities on ecological flow assurance based on a complete set of frameworks. The present study introduces an integrated assessment framework designed to quantify the impacts of climate change and human activities on ecological flow security. The framework includes the following steps: (1) natural river runoff reconstruction utilizing a semi‐distributed hydrological model (SWAT), (2) calculation of the most suitable ecological stream flow of the watershed ecosystem by using the non‐parametric kernel density estimation method, (3) calculation of the safety and security levels under minimum ecological flow and appropriate ecological flow conditions in the watershed and (4) quantification of the influences of climate change and human activities on the security of ecological flow in the watershed through the application of a quantitative attribution method. The impact of climate change and human activities on the ecological flow assurance level was analysed using three hydrological stations in Xiangtan, Hengyang and Laobutou, which are the main tributaries of the Xiangjiang River Basin, as a case study. The findings indicated a substantial decrease in ecological flow assurance levels across the basin during the period of human impact (1991–2019). The quantitative assessment results suggest that human activities predominantly drive the degradation of ecological flow assurance throughout the period of human impact, accounting for 57.05% of the total impact. Extensive gradient reservoir scheduling and anthropogenic water withdrawals were the main factors contributing to the degradation of ecological flow assurance in the study basin. The methodology and findings presented in this study offer insights into the evolutionary characteristics and driving forces behind ecological flow security in a dynamic environment. Furthermore, they establish a scientific foundation for local water resource management and river ecosystem protection.