The water conveyance pipeline project plays an important role in solving the supply and demand contradictions and uneven geographic distribution of water in China. Considering the water conveyance pipeline project from a socially beneficial perspective, water supply would increase after the project completion in northern China, where there is heavy water scarcity; water environmental carrying capacity in these regions would be enhanced as well. From an economically beneficial perspective, construction of the pipeline for the water conveyance project would help to increase the regional output values of industry and agriculture, as well as create more jobs for the local residents and others. From an eco-environmentally beneficial perspective, the water conveyance pipeline would provide new solutions to problems such as groundwater overdraft and water pollution in the water-recipient regions. However, even with the aforementioned social, economic, and eco-environmental benefits to relevant regions, the construction of the water conveyance project also creates new problems for the fragile ecosystem and environment such as disruption of the ecosystem balance in the water source area, damage of the phreatic aquifer along the pipeline project, and so on. Therefore, we need to invest more funds, technology, and personnel into the construction and management of the water conveyance project because of this duality. At the same time, there are many obstacles to an ecological impact assessment of water conveyance projects, one of the most important being the lack of quantitative assessment methods. The determination of assessment scope, lack of ecological monitoring and feedback activities are also significant issues to consider in ecological impact assessments. How to assess the eco-environmental impact of water conveyance projects scientifically and quantitatively is a key decision to project construction and management. Initially, when the concepts of ecological water requirements were proposed in 1998, many researchers from various limnological disciplines of study, such as rivers, lakes, and wetlands, paid close attention to these concepts and relevant methods. Researchers interpreted and analyzed the concepts of ecological water requirements from hydrology, hydraulics, and ecosystem perspectives. The assessment methods and calculation models of ecological water requirement were enriched gradually, and the typical methods, such as Tennant, R2CROSS, and IFIM, among others, were widely applied in the assessment of ecological water requirement of rivers, lakes, and wetlands. In this study, based on the assessment theory, methods of ecological water requirement, and the quantitative analysis of ecological impact assessment, further reinforcing the relationship between ecological water requirement and ecosystem health, we established a technological roadmap and an estimation model for the eco-environmental impacts of the water conveyance project, which lie within the ecological water requirements. Using the Middle Route of the South-to-North Water Division Project as a case study, we assess the ecological water requirements of the relevant influenced regions. The results show that water transfer during the first phase of the South-to-North Water Division Project would not have a serious impact on water resources and the ecosystem in the region and would not result in the obvious ecosystem degradation. Additionally, the results prove that methods of ecological water requirement assessment are helpful to ecological impacts assessment.