In order to solve the problems such as water resources shortage, excessive water loss, low water use efficiency, agricultural irrigation water squeezes ecological water, based on the water control policy of “water conservation priority”, the mountainous reservoirs and plain reservoirs in the two source streams of the Tarim River (Aksu River, Yeerqiang River) and its main stream (“two source streams and one main stream”) are taken as the research objects. Considering the multiple objectives of agricultural irrigation water, ecological water, industrial water and domestic water in the mainstream of Tarim River, an optimization model for joint operation of reservoirs in mountainous areas and plains in the current level year and the long-term planning year is constructed and solved, and the ecological water supply potential of the mainstream of Tarim River and the contribution of joint operation of reservoirs in mountainous areas are analyzed. The results show that the guarantee rate of agricultural irrigation and ecological water supply cannot meet the design requirements only through the joint operation of reservoirs in the mainstream plain. Through the optimal operation of reservoir groups in mountainous areas and plain areas, all industries meet the design assurance rate requirements in the long-term planning year, reflecting the remarkable regulation performance of reservoirs in mountainous areas and the rationality and reliability of the water control policy of "water saving first". The ecological water supply potential of the mainstream of Tarim River in the long-term planning year is 284 million m³, which can provide a solid water volume basis for ecological protection and restoration in the lower reaches of the Tarim River.