Inter-basin water transfer (IBWT) projects are an effective means of addressing regional water resource imbalances. However, owing to the long construction cycle, large investment amount, and wide impact range, water diversion projects exhibit delayed and complex benefits, often lacking clear comprehension. In this study, we established a multi-regional comprehensive benefit assessment framework for the IBWT, considering spatiotemporal and multi-dimensional value effects. Using the South-to-North Water Diversion Project (SNWDP) in China as an example, we assessed its comprehensive benefits from 2003 to 2020. The results showed that the comprehensive benefits of the project were USD 207 billion, encompassing economic and ecological benefits, accounting for 71.6% and 28.4%, respectively. In 2020, the benefits of the SNWDP amounted to USD 39.3 billion, with a per-unit area benefit range of USD −1.03 to 4.27. The operation of the SNWDP effectively alleviated groundwater overexploitation in water-receiving areas. However, without the SNWDP, the total loss caused by industrial development limitations due to water scarcity would have been USD 154.3 billion. These results indicate the importance of a practical framework for assessing IWBT project benefits, aiding managers in assessment tasks, and facilitating the prediction and adjustment of project benefits.