Abstract: The Mekong river basin is bringing great economic and ecological values in the world, expecting to support the scheme of China’s Belt Road Initiative. Taking the sharing of Mekong River Basin as a case study, this study aims to calculate the Ecosystem Service Value (ESV) and compensation benefits for countries in the Mekong River Basin, in order to maintain the linkage between benefit sharing and economic compensation in Mekong River Basin. A biophysical approach based on the Ecological Footprint (EF) was used to measure the ESV and ecological surplus (or deficit) in the Mekong River Basin in 1995 and 2015. The relationship between ESV and actual compensatory payment for ecosystem services was initially investigated to quantitatively analyze the priority of ecological compensations. The results showed that: 1) The ESV decreased 3 billion dollars (from 128.976 to 125.921 billion dollars) during 1995-2015 in the Mekong River Basin. Specifically, Thailand presented the maximum ESV, followed by Laos, Cambodia, Vietnam, and Myanmar. The qualitative assessment revealed that the highest levels of ESV were derived from forest in every country (>61.7%). There was a 4.5% increase in the ecosystem service that provided by water provisioning and wetlands in the past 20 years. 2) Myanmar and Laos were in the state of “ecological surplus”, while the other three countries were in the state of “ecological deficit”, indicating an increase deficit level. Thailand and Vietnam showed more than 80.1% of the total ecological footprint (EF) of the basin. 3) Myanmar and Laos were also the “ecological export” countries, whereas, the three countries in the lower reaches were the “ecological consumption” countries. Thailand and Vietnam can give a priority on the payment for ecosystem services because of its better economic outcomes and lower Ecological Compensation Priority Sequence (ECPS) values of 0.05 and 0.09, respectively, indicating that lower than the other three countries in the Mekong River Basin (Myanmar: 2.67, Laos: 1.16, Cambodia: 0.55). 4) In the course of economic growth of the countries in Mekong River Basin, the total ecological compensation required 68.063 billion dollars from the “ecological consumption” countries in the lower reaches. Thailand, Vietnam, and Cambodia can be required 50.773, 16.761 and 0.529 billion dollars, respectively. This finding can provide a theoretical support to establish the policies for resource management and compensatory payment in the Mekong River Basin and other transboundary river basins.