Understanding the response of long-term aquatic environmental changes in lakes to ongoing climate change and human activities is key to forecasting future lake conditions. In this study, we infer the Holocene limnological changes in Emu Co, a proglacial lake in Nianbaoyuze on the eastern Tibetan Plateau, from sedimentary ancient DNA (sedaDNA) data, and palynomorph, element, lithological, and grain-size analyses. We developed a transfer function based on Siberia and Tibet/China surface sedimentary DNA and applied it to Emu Co sedaDNA to trace lake conductivity changes. The results show that the conductivity of Emu Co was high during 12.6−9.7 cal ka BP, often surpassing 1000 μs cm−1, driven by elevated summer solar radiation. The freshwater influx from glacial meltwater and precipitation, however, reduced the lake's conductivity as the climate warmed and humidified. This led to a decrease in the abundance of taxa characterised by high conductivity. Freshwater pulses, triggered by climatic fluctuations, likely led to significant variations in conductivity within the overarching downward trend. By 8 cal. ka BP, lake recharge conditions stabilised and conductivity reached a lower level of ∼70 μs cm−1. The warm and humid mid-Holocene (8−5 cal. ka BP) provided suitable habitat conditions for many submerged freshwater taxa. After 5 cal. ka BP, the growth of submerged taxa was restricted, as indicated by a shift from asexual to sexual reproduction in macrophytes, likely in response to suboptimal conditions of a colder and drier climate. Since 1 cal. ka BP, human activities might have increased lake nutrient levels, with an enhanced richness of macrophytes. Our results indicate how millennial-scale hydrological changes in a lake are related to glacial retreat and catchment changes in the alpine region of the Tibetan Plateau, which is today facing climate change much greater than the global average.