The Middle Route of the South-to-North Water Diversion Project (MRSNWDP) is the most important inter-basin water diversion project in China so far, and provides the scarce production and domestic water for 24 large and medium-sized cities along the route. However, the dynamic change process of the ecosystem service value of the project was studied so inadequate that hindered the scientific evaluation of the project ecological benefits and the rational use of land resources. The study on changes in the value of ecological services before and after the completion of the MRSNWDP is helpful to the rational planning and scientific use of land along the route. This paper analyzed the temporal and spatial changes of the static value of ecological services of the project in 2000, 2005, 2010, 2015 and 2018 based on the improved equivalent factor method, and then reconstructed the dynamic value of its ecological services with the priority index, and discussed its temporal and spatial evolution characteristics. The results showed that:① from 2000 to 2018, the total value of ecological services in the study area first decreased and then increased, and reaching its peak in 2018. The increase in the value of ecological services in the study area can be attributed to the impact of the MRSNWDP to a certain extent. ② The value of the hydrological regulation function was the highest, accounting for 30% of the total value, and that of the nutrient cycle function was the lowest, accounting for only 1%. The forest land, grassland, cultivated land and water body were the main components of the total value of ecological services in the study area in each year. ③ The spatial distribution of ecological service values in the study area was significantly different from 2000 to 2018, which decreased from the west to the east. The high-value areas in Beijing and Tianjin increased after water diversion. ④ The total dynamic value of ecological services in the study area was 1.38×10¹² Yuan, which was lower than the static total value of 2.58×10¹² Yuan, due to the value coefficient of dynamic and static ecological services is quite different. Therefore, we should pay attention to the impact of changes in land use types and weak ecological service functions on the total value of ecological services, optimize the structure of land use, and carry out targeted protections along the MRSNWDP to steadily improve the total value of ecological services in the entire region.