Qilian Mountains are important ecological security barrier and priority area for biodiversity protection in western China, of which the southern slope area is the core area for water conservation services. However, at present, there is still a lack of in-depth understanding of the temporal and spatial distribution and dynamic changes of ecosystem services in this region. This paper used remote sensing technology and ecosystem service theory, and integrated multi-source data products and ecosystem service calculation methods to monitor and evaluate the spatio-temporal variation and dynamics of the values of seven ecosystem service types in the southern slope of Qilian Mountains (SSQM). The results showed that (1) in the past 16 years, the average unit area value of ecosystem service on the SSQM was 762700 yuan/km², with a total value of 65.87 billion yuan. The climate regulation, water conservation, and air quality regulation services were the dominant service types. (2) The alpine meadow and alpine steppe had the largest total ecosystem service value (ESV), while forest and shrubland had the largest service value per unit area. Within the study area, the total ESV of Tianjun County and Qilian County was the largest, while the unit area value in Haiyan County and Gangcha County was the largest. (3) In the past 16 years, the ESV on the southern slope of Qilian Mountains has shown an increasing trend, of which the service value of climate regulation and air quality regulation had the fastest growth rate. The growth areas were mainly concentrated in Qilian County and Tianjun County in the northern part of the research area. (4) The mountain area in the east, the Qinghai lake belt in the south and the wetland area in the center can provide multiple composite ecological service functions and should be taken as the focus of the ecological protection and management. The results can provide the theoretical basis and technical support for establishment of Qilian mountain ecological red line and the construction of national parks.