Abstract A thorough understanding of the ecological impacts behind the hydrologic alteration is still insufficient and hinders the watershed management. Here, we used eco-flow indicators, multiple hydrological indicators, and fluvial biodiversity to investigate the ecological flow in different temporal scales. The case study in the Han River shows a decrease in high flows contributed to the decrease in eco-surplus and increase in eco-deficit in summer and autumn, while the decrease in eco-deficit can be attributed to the change of low flow in spring. An integrated hydrologic alteration was over 48% degree and was under moderate ecological risk degree in impact period I, while DHRAM scores showed the Huangzhuang station faced a high ecological risk degree in impact period II. The decrease (increase) in total seasonal eco-surplus (eco-deficit) was identified after alteration with the change in seasonal eco-flow indicators contributions. Shannon index showed a decreasing trend, indicating the degradation of fluvial biodiversity in the Han River basin. Eco-flow indicators such as eco-surplus and eco-deficit are in strong relationships with 32 hydrological indicators and can be accepted for ecohydrological alterations at multiple temporal scales. This study deepens the understanding of ecological responses to hydrologic alteration, which may provide references for water resources management and ecological security maintenance.