<p>Reservoir operation and water division have significant altered the downstream hydrologic regime and ecological environment. The ecological problems, such as algal blooms, invasive alien species, and fish reproduction, in the middle and lower reaches of Han River basin in China is becoming prominent in recent years. The objective of this study is to propose a multi-objective optimal operation scheme that simultaneously considers the hydrologic alternation and ecological water demand. Based on the Indicators of Hydrologic Alteration (IHA), the Revised Range of Variability Approach (RRVA) was used to estimate the change of hydrologic regime. Two new ecological indexes, i.e., Water Quantity Level (WQL) and Hydrologic Alteration (HA), were also established. The ecological water demand in the downstream was determined and set as the constrain of outflow discharge in the reservoir operation. Four objectives (water supply, power generation, WQL and HA) operation model of the Danjiangkou Reservoir was established and solved by the Non-dominated Sorting Genetic Algorithm II, in which the Gaussian Radial Basis Functions (GRBFs) were constructed to fit the reservoir operation rules. The results revealed that overall degree of hydrological alteration in the downstream is 62.38% for Han River basin. There are conflicts between ecological indexes and the water supply. Compared with current standard operation scheme, the multi-objective operation scheme considering ecological flow could improve water supply (12.12%) and power generation (1.68%), and achieve the optimal effect of WQL and HA by adjusting the outflow discharges. Meanwhile, the optimal operation scheme could arise the comprehensive ecological index by 10.21% and meet the ecological water demand to restrain water ecological problems in the downstream. The proposed method may provide guidance for multi-objective reservoir operation and ecological protection in the Han River basin.</p>