Sediments from Tibetan lakes in NW China are potentially sensitive recorders of climate change and its impact on ecosystem function. However, the important plankton members in many Tibetan Lakes do not make and leave microscopically diagnostic features in the sedimentary record. Here we established a taxon-specific molecular approach to specifically identify and quantify sedimentary ancient DNA (sedaDNA) of non-fossilized planktonic organisms preserved in a 5-m sediment core from Kusai Lake spanning the last 3100 years. The reliability of the approach was validated with multiple independent genetic markers. Parallel analyses of the geochemistry of the core and paleo-climate proxies revealed that Monsoon strength-driven changes in nutrient availability, temperature, and salinity as well as orbitally-driven changes in light intensity were all responsible for the observed temporal changes in the abundance of two dominant phytoplankton groups in the lake, Synechococcus (cyanobacteria) and Isochrysis (haptophyte algae). Collectively our data show that global and regional climatic events exhibited a strong influence on the paleoecology of phototrophic plankton in Kusai Lake.