[Objective] This study aimed to establish a method for calculating horizontal ecological compensation standards for the Yangtze River Basin based on an assessment of water resource utilization in various regions. Using the years 2010, 2015, and 2020 as examples, it sought to mitigate conflicts arising from the development and utilization of water resources among provinces in the basin through ecological compensation mechanisms, thereby promoting the protection of water resources, the restoration of aquatic ecosystems, and the sustainable development of the regional economy and society. [Methods] The research first calculated the value of ecosystem services provided by water resources in the Yangtze River Basin using water surface area and a modified equivalent factor table. Second, the study calculates the freshwater and water pollution ecological footprints and ecological carrying capacity across the regions of the basin to evaluate their water resource overload status. Finally, using the watershed-wide water resource ecological overloading coefficient as a baseline, the identities of the compensation subjects and objects in each region were defined. Based on the theory of public goods attributes and externalities, the theoretical amount and the lower limit value of ecological protection compensation in each region are calculated by combining the water resource ecosystem service value. [Results] ①From 2010 to 2020, the value of ecosystem services from water resources in the Yangtze River Basin increased annually, amounting to 11693.50 billion, 11785.48 billion, and 11915.91 billion yuan, respectively. ②Between 2010 and 2020, the freshwater ecological footprint of the Yangtze River Basin first increased and then decreased, while the water pollution ecological footprint continued to rise. Based on the concept of ecological carrying capacity, the basin did not experience overload during the study period. However, there were water supply-demand gaps in regions such as Shanghai, Jiangsu, and Henan. ③During the study period, Qinghai, Tibet, Guangdong, Guangxi, Hunan, Jiangxi, and Fujian were consistently the ecological protection compensation recipients, while Anhui, Yunnan, Zhejiang, Henan, Jiangsu, and Shanghai were consistently the compensation payers. The compensation amounts paid by the payer regions were within the scope of their general fiscal budgets and showed a year-on-year decline. [Conclusion] This study developed a cross-regional water resource horizontal ecological protection compensation calculation model for the Yangtze River Basin, which not only defines the compensation subjects and objects but also clarifies the flow of compensation funds. This model helps stimulate active participation from various regions and holds significant importance for promoting the rational use of watershed water resources and ensuring social equity.