At present, nearly half of the population of China live in water-deficient areas where water needs to be transferred from surrounding or remote water sources to meet local water demands. Although the water transfer project has alleviated the demands for water in the water-deficient areas, and brought water-supply income to water source regions, it has also posed some cross-regional negative impacts, including the changes in the original ecology within the water source, the impacts on the downstream water demands, and the risk of biological invasion in the distant water receiving areas. Therefore, it can be seen that the impact of water transfer is complicated and will be manifested in various aspects. The Middle Route of China’s South–North Water Transfer Project (SNWTP-MR), as the world’s largest cross-watershed water transfer project, exerts particularly important effects on regional sustainable development; however, it also produces complex interactions within the ecological environment itself, downstream and in the distant water receiving cities. Thus, this work attempts to apply a metacoupling analysis framework of water transfer to explore the ecological interaction of water transfer in SNWTP-MR on each system. The metacoupling framework can be divided into intracoupling, pericoupling and telecoupling. This study focuses on the analysis of the causes and effects of the intracoupling, pericoupling and telecoupling of SNWTP-MR from the perspective of ecological values and ecological risks. We found that the coupling of water transfer brings about 23 billion yuan of ecological service value to the water source annually, but also increases the internal ecological risk index by 9.31%, through the calculation of changes in land use; secondly, the power generation benefit significantly increases, and the flood control standards have shifted from once-in-20 years to once-in-a-century. However, the ecological risks are also significant, such as poor water quality, eutrophication of water resources, competition for water between industry and agriculture, deterioration of waterway shipping, and threats to biodiversity, etc. Considering only water supply, the population carrying capacity of the water resource in distant water receiving cities is increased by 16.42 million people, which enhances the value of water resources and creates a cross-regional green ecological landscape belt. Nevertheless, the biological invasion and water pollution have greatly affected the safety of water supply. It can be seen that the cross-regional water transfer does not always damage the interests of the sending system and the spillover system while benefiting the receiving system; its impacts are complex and variable. Through this paper, it is hoped to provide a reference for the analysis of the ecological compensation, resource development and allocation in SNWTP-MR by revealing the metacoupling relationship of SNWTP-MR. This paper will provide new ideas for researching the metacoupling relationship, thereby offering valuable reference for the study of the interaction generated by large-scale water transfer.