The ecological environment protection of the Qilian Mountains is the key process in the construction of ecological safety barriers in West China. Calculation of ecosystem service values in the Qilian Mountains is the strong basis for formulating reasonable and effectively ecological environment protection measures. However, due to the joint impact of climate change and human activities, the ecological and environmental problems in the Qilian Mountain region are prominent, and it is urgent to carry out research on the ecosystem service values in this region to serve the later ecological and environmental construction. This study used the land-use data from 1990 to 2020 to reveal the variation trend of land-use change in the Qilian Mountain region by using the land-use dynamic attitude and land-use transfer matrix. Furthermore, the ecosystem service value in the Qilian Mountain area was estimated by using the ecosystem service value assessment model, and its dynamic change process was explored. Additionally, the internal relationship between each land-use type and the ecosystem service value changes was clarified. The results showed that: (1) From 1990 to 2020, grassland and desert were the most dominant land-use types in the Qilian Mountains, and the single land-use dynamic degree was water area > wetland > cultivated land > desert > grassland > forest land. The interconversions of grassland with cropland and desert were the main features of land-use change in the region. (2) The values of ecosystem services in the Qilian Mountains region showed a continuous growth trend on the time scale, from 723.136 billion yuan in 1990 to 783.607 billion yuan in 2020, with a spatial trend of high in the east and low in the west. The main components of ecosystem service values were water body area, grassland and forest land. The value of individual ecosystem services was dominated by hydrological regulation and climate regulation, reflecting the fact that regulation services were the main ecosystem functions in the Qilian Mountains. (3) Water area, grassland and wetland were the main factors contributing to the change of ecosystem service value in the past 30 years. From 1990 to 2020, the sensitivity of ecosystem service value to land-use showed an overall increasing trend (1.48%-7.91%), and the impact of land-use change on ecosystem service value gradually increased. The study systematically revealed the evolution of ecosystem service value and its internal relationship with land-use change in the Qilian Mountain region in the past 30 years and highlighted the importance of water, grassland, forest land and wetland to ecosystem service value and ecological environmental protection in the region. It can provide basic scientific and technological support for ecological product value implementation and ecological protection management in the Qilian Mountain region.