This study aimed to evaluate the ecohydrological regime and ecological water demand of the Huangshui River Basin under changing environmental conditions, seeking to safeguard its ecosystem. Based on monthly data spanning from 1956 to 2016, the ecohydrological regimes of the Huangshui River and the Datong River were evaluated using methods such as the Pettitt mutation test, the Tennant method, and ecological deficit and surplus analyses. The data were mainly obtained from Xiangtang Station of the Datong River and Minhe Station of the Huangshui River. The results showed the following. (1) The most abrupt increase in measured runoff at Xiangtang Station occurred in 1993, while the point of abrupt change in measured runoff at Minhe Station occurred in 1990. (2) Following an increase in human activities, changes in the ecological surplus at Xiangtang Station were negative in January, April to May, July, and from September to November, while the changes in the ecological deficit were positive from January to April, July to August, and October to December. Changes in the ecological surplus at Minhe Station were negative from March to July and from September to December, while changes in the ecological deficit were positive from January to April and from July to December. (3) The annual average ecological flow of the Datong River, Xiangtang section, was 28.42 m3/s, and the annual average ecological water demand was 896 million m3. The annual average ecological flow of the Minhe section was 19.98 m3/s, and the annual average ecological water demand was 631 million m3. According to a calculation of the degree of ecological water demand and ecological flow satisfaction, prior to the implementation of the Water Diversion Project from the Datong River to Huangshui River, the water volumes in both rivers were generally sufficient to meet the ecological water demand. However, high water consumption during the irrigation period led to an ecological deficit. To address these issues, it is crucial to evaluate the potential impacts of human activities, such as water diversion projects, on river ecological flow. Recommendations include expediting the Water Diversion Project from the Yellow River to Xining to secure sufficient water flow in the Huangshui River and enhancing water conservation efforts in agricultural irrigation.