Hydrological regulation is one of the services provided by watershed ecosystems. For watershed ecosystem conservation and restoration and reasonable development of watershed water resources, it is important to identify the spatial pattern of watershed ecosystem hydrological regulation service and its driving forces. Many studies have been conducted at the ecosystem and landscape scale on the influence of different vegetation types and landscape patterns on hydrological regulation; such studies have played an important role in furthering the understanding of the processes and mechanisms of the hydrological regulation service in the ecosystem. At the basin scale, hydrological regulation services were affected comprehensively by many factors, including ecosystem characteristics, water conservancy facilities, water used by humans, and so on, and these influences have scale effects. Presently, owing to the lack of comprehensive studies of the systematic ecosystem at the river scale, it is still difficult to evaluate ecosystem effects on regional hydrological processes from a macro perspective. In addition, few studies have explored how to represent and understand ecosystem water regulation services at the river scale as well as to identify influential factors that have differing effects on the water regulation service of the ecosystem at the river scale. Thus, a study on the space variation regularity of hydrological regulation services and its diving force is necessary. We used as a case study the Yangtze River Basin, where flooding occurs frequently, as to study the spatial patterns of ecosystem hydrological regulation services and factors affecting it, by using the variation coefficient method and multi-variable statistics methods. The results showed that, in the Yangtze River Basin, ecosystem hydrological regulation services presented significant spatial heterogeneity at the sub-watershed scale. The sub-watersheds in Jinshajiang and Hanjiang watersheds, where dense hydropower engineering facilities are located and the coverage of natural vegetation is 71%, had the highest regulating capacity of hydrology. The difference between the coefficients of variance of precipitation and runoff was 0.477. In contrast, sub-watersheds with high population density and high proportion of cropland in Jialingjiang watershed had the lowest regulating capacity of hydrology and the difference between the coefficients of variance of precipitation and runoff was -0.474. The factors primarily affecting sub-watershed ecosystem hydrological services include reservoir volume, natural vegetation proportion, cropland proportion, and population density. Reservoir volume and natural vegetation proportion had positive effects on sub-watershed ecosystem hydrological services, while cropland proportion and population density had negative effects. Reservoir volume had the highest contribution to the heterogeneity of sub-watershed ecosystem hydrological services (58.85%). Our results facilitate a better understanding of the spatial heterogeneity of sub-watershed ecosystem hydrological services in Yangtze River Basin, and could also provide a scientific basis for sub-water ecosystem conservation and restoration, as well as the improvement of sub-watershed ecosystem hydrological regulation services.