Ecosystem services (ESs) are an important bridge coupling the natural environment and human society. The Qinghai-Tibetan Plateau (QTP), as an ecological barrier in China and even in Asia, plays a key role in maintaining regional ecological security and sustainable supply of water resources. However, there is limited research on determining thresholds and zoning for the effects of fractional vegetation cover (FVC) on ESs over long periods of time. This study took the QTP as the study region, analyzed the long-term changes and short-term fluctuations of key ESs, determined the impact thresholds of FVC, and designated the ecological functional areas of the QTP and different land types. The results showed that all key ESs increased of different degrees from 2000 to 2022. The mean values of the Interannual Fluctuation Index (IFI) for Water Yield (WY), Habitat Quality (HQ), Carbon Storge (CS), Soil Conservation (SC), Climate Regulation (CR) and Food Supply (FS) were 13.08%, 5.66%, 36.01%, 8.31%, 22.77% and 51.69%, respectively. The impact thresholds of FVC on Total Ecosystem Services (TES) in the QTP, grassland, forest and barren were 0.44, 0.40, 0.29 and 0.23, respectively. Based on the threshold analyzed, the Ecological Safety Area (ESA), Ecological Transition Area (ETA) and Ecological Protection Area (EPA) were delineate, which provided theoretical support for the ecological restoration and sustainable development of the QTP.