The Qinghai Lake basin (QLB) is an ideal region to explore the interaction between climate change, vegetation evolution, and anthropogenic activities during the Holocene epoch. This is due to the basin’s particular sensitivity to global climate changes along with its densely distributed archeological sites, present since the last deglaciation. To date, a full understanding of the lakeshore terrestrial fossil pollen record is lacking and few studies have explored the human signals in the pollen spectra of the northeast Qinghai-Tibetan Plateau. Here, we present fossil pollen and charcoal records from over 8500 years ago obtained from a high resolution-dated aeolian section in the southeastern margin of the QLB. These results show that the pollen spectra are dominated by herbaceous and shrubby pollen taxa, while arboreal pollen taxa are rare. From approximately 8.5–7.2 ka, Artemisia- and Poaceae- dominated temperate steppe developed in the region, suggesting relatively warm and dry climatic conditions. Comparatively, regional vegetation shifted to Cyperaceae-dominated alpine meadow from approximately 7.2–3.4 ka, which reflected cooler and wetter climatic conditions. Thereafter, the amount of Asteraceae, Artemisia, and Chenopodiaceae notably increased from approximately 3.4–1.8 ka, suggesting that temperate steppe dominated the region under relative drier climate. Noticeably, an interval between approximately 1.8–0.6 ka was characterized by an increase of Hippophae pollen, reflecting a comparatively warmer episode; alpine meadow then reoccupied the region since approximately 0.6 ka. Combining the variations of charcoal concentrations (20–50 µm and >50 µm) with human-related pollen taxa (Aster-type, Chenopodiaceae, Plantago, and Hordeum-type), we inferred that prehistoric humans were extensively using fire for hunting and subsistence during 7.2–3.5 ka, which strongly impacted the local vegetation during the middle Holocene; regional grazing activities intensified and caused grassland degradation since approximately 3.5 ka, which manifested with a significant increase in Aster-type and Chenopodiaceae pollen. After this period, Hordeum-type pollen as well as charcoal concentration of >50 µm increased significantly since approximately 2.2 ka, suggesting an intensifying agricultural cultivation in the region.