The runoff change in the middle and upper reaches of the Hutuo River was analyzed, and ecological flow of the river was calculated. A multi-objective optimization model for water supply and ecological operation of Gangnan-Huangbizhuang cascade reservoirs was established, and NSGA-II was used to solve different schemes in various scenarios. The results showed that the runoff of Xiaojue and Pingshan stations had a significant decreasing trend, and changed abruptly in 1983 and 1979, respectively; annual ecological water demands corresponding to the optimal, suitable, and minimum ecological flows of the river, calculated with the optimal distribution function for each month, Tennant method, and dynamic calculation method, were 985 million, 585 million, and 323 million m³, respectively. Reservoir operation results also showed that the water shortage rate of agricultural irrigation and ecological water demands increased significantly with the increase of the inflow frequency, and the water supply guarantee rate under reservoir operation in different level years decreased due to the significant decrease and sudden change of water inflow. In addition, there is a significant contradiction between irrigation water demand and ecological guarantee, and reducing the rate of water supply for irrigation by 4% may increase the ecological water supply rate by 12% in dry years.