An ecosystem’s flood regulation service (EFRS) is important for alleviating flood risk. Both the EFRS and the relationship between EFRS supply and demand may change across flood return periods. However, most studies only considered the EFRS under a single flood return period. In this regard, a new framework is proposed that integrates multiple flood return periods to assess the expected annual EFRS supply and demand. It also considers the upstream EFRS supply flowing from the headwaters toward the downstream. A case study in the Yangtze River Basin, China, shows that the EFRS demand increases dramatically from 25- to 200-year return periods, while the EFRS supplies remain relatively constant. In terms of the expected annual EFRSs that integrate the four return-period EFRS demands and local supplies, 47 (or 43.12%) sub-basins are found to be unsatisfied (the demand is larger than the supply). This number could be reduced to 17 (or 15.60%) sub-basins if the upstream EFRS supply is considered in comparing the EFRS supply and demand, implying the importance of a catchment-wide EFRS management, particularly protecting the upstream forests, rivers and lakes, grasslands, as well as properly regulating the dams. The proposed framework could be readily applied in other basins and shed light on a comprehensive understanding and management of the EFRSs.