Abstract: The Yellow River is the second largest river in China and is the important source for water supply in the northwestern and northern China. It is often regarded as the mother river of China. Owing to climatic change and intensifying human activities, such as increasing withdrawal of water for meeting growing agricultural irrigation needs since 1986, the flow of Yellow River has decreased, with serious impacts on the ecological environment. Using multiple hydrological indicators and Flow Duration Curve (DFC)-based ecodeficit and ecosurplus, this study investigates the impact of hydrological alterations, such as the impact of water reservoirs or dams, on downstream ecological instream flow. Results indicate that: (1) due to the impoundment and hydrological regulations of water reservoirs, occurrence rates and magnitudes of high flow regimes have decreased and the decrease is also found in the magnitudes of low flow events. These changes tend to be more evident from the upper to the lower Yellow River basin; (2) human activities tend to enhance the instream flow variability, particularly after the 1980s;(3) the ecological environment in different parts of the Yellow River basin is under different degrees of ecological risk. In general, lower to higher ecological risk can be detected due to hydrological alterations from the upper to the lower Yellow River basin. This shows that conservation of ecological environment and river health is facing a serious challenge in the lower Yellow River basin; (4) ecological instream flow indices, such as ecodeficit and ecosurplus, and IHA32 hydrological indicators are in strong relationships, suggesting that ecodeficit and ecosurplus can be regarded as appropriate ecological indicators for developing measures for mitigating the adverse impact of human activities on the conservation of ecological environment in the Yellow River basin.