Abstract: The quantitative evaluation of ecosystem services is the basis of ecosystem services management, which is of great significance to the construction of ecological civilization, such as asset management of natural resources and ecological compensation. Three-River Headwaters Region (TRHR) is an important ecological security barrier in China. The evaluation of ecosystem services in this region and its value clarification are helpful to improve their ecological environment protection. Based on satellite remote sensing images, ground-based ecological monitoring data, statistical data, The TRHR’s ecosystem services such as hydrological regulation, water supply, soil conservation, ecological carbon sequestration and species conservation were assessed over the period from 2000 to 2015. The results showed that in the past 15 years, with the increasing efforts of the ecological environment protection and construction, its ecosystem services had been improved as a whole. Ecosystem service value increased from 480.558 billion yuan in 2000 to 537.979 billion yuan in 2015, with an increase of 12.5%. The ecological service value per area was 1.22 million yuan/km² in 2000, 1.23 million yuan/km² in 2005, 1.41 million yuan/km² in 2010 and 1.36 million yuan/km² in 2015, respectively. Compared with 2000, the ecosystem services value of about 55.5% area of TRHR increased significantly in 2015, while about 42.4% area had no obvious change and only about 2.1% area decreased significantly.