Quantitative reconstruction of paleotemperature has become a critical link in understanding the evolution of the Earth’s climate system. However, only few reliable continental temperature reconstruction indicators are available. Glycerol Dialkyl Glycerol Tetraether membrane lipids (GDGTs) are derived from microbial cell membranes. The MBT/CBT and TEX86 indices related to GDGTs have been applied to the quantitative paleotemperature reconstruction of lake systems. However, because of the complexity of the lake systems and the particularity of the plateau climate, the application of relevant indicators in lakes on the Qinghai-Tibet Plateau is affected. Therefore, it is vital to conduct detailed modern process analyses of the target lakes and investigate the potential applications of these indices. In this study, 24 samples of soil, lake, and river surface sediments were collected from Heihai Lake on the northern Tibetan Plateau. The sources of GDGTs in the Heihai Lake sedimentary system were analyzed. The responses of the GDGTs and their related indicators to environmental factors were discussed. The applicability of the MBT/CBT index and temperature conversion function to the temperature reconstruction of plateau lakes was investigated. Results show that there are some endogenous GDGT compounds present in Heihai Lake, and that water depth has a significant effect on the composition and distribution of GDGTs. Conductivity and TOC have weak effects on GDGTs, and the MBT/CBT index has some limitations in reconstructing lake temperature in high-altitude and cold regions. Therefore, more suitable temperature indicators and correction equations for plateau lakes need to be established by combining the regional characteristics, climate types, and ecological responses of microorganisms to special environments.