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Based on the meteorological data, remote sensing data, soil types, geological background and socio-economic data of Chishui River Basin in 2000, 2010 and 2015, the total value of ecosystem services in the basin was quantitatively assessed by carbon sequestration and oxygen release model, modified general soil loss equation (RUSLE) and InVEST water conservation model. At the same time, the ecological compensation quota of different regions in the basin was calculated by using the eco-compensation econometric model, and the compensation standard and priority of the basin were determined. The results indicated that from 2000 to 2015, the value of carbon sequestration and oxygen release per unit area, the value of soil conservation and value of water conservation in Chishui River Basin showed an inverted “V” structure. The value of carbon sequestration and oxygen release was increased from 4279.33 yuan/(hm2·a) in 2000 to 4520.16 yuan/(hm2·a) in 2010, and then decreased to 4409.69 yuan/(hm2·a) in 2015;the value of soil conservation was increased from 95.11 yuan/(hm2·a) in 2000 to 144.45 yuan/(hm2·a) in 2010, and then decreased to 89.03 yuan/(hm2·a) in 2015;and the value of water conservation was decreased from 23938.13 yuan/(hm2·a) in 2000 to 35232.99yuan/(hm2·a) in 2010, and then decreased to 20167.41 yuan/(hm2·a) in 2015. The total value of carbon sequestration and oxygen release in karst area was increased by 4.35 times as fast as that in non-karst area, while the value of soil conservation and water conservation in karst area was decreased by 1.28 and 1.59 times as fast as that in non-karst area, respectively. The value of ecological compensation per unit area was 4626.29 yuan/(hm2·a), and the average value of ecological compensation was 7.623 billion yuan. The value of compensation in the lower reaches of the basin was the largest, while that in the upper reaches was the smallest. The karst area in the upper reaches of the river basin and the primitive forest area in the lower reaches belonged to the typical ecological export area, which should be compensated first. The economically developed middle reaches of the river basin had active economic development level and should take the lead in ecological payment, which belonged to the typical ecological consumption area. Compared with the previous ecological compensation model based on value equivalence, the research quantified the ecological compensation standards of different areas in the basin on grid pixel scale, which can provide scientific basis for the ecological compensation model, operation mechanism and compensation standards of Chishui River Basin in the upper reaches of the Yangtze River.