The relationship between groundwater and lakes in Qaidam Basin is often overlooked. Therefore, we employed Landsat satellite images and meteorological data to investigate the causes of lake expansion through model calculation and statistical analysis and then determine groundwater sources through isotope analysis (²H, ³H, and ¹⁸O). In the two study periods of 2003–2011 and 2011–present, temperature, precipitation, and runoff increased at a steady rate, whereas the expansion rate of Tuosu Lake increased from 1.22 km²/yearr to 3.38 km²/yearr. This significant increase in the rate of lake expansion reflects the substantial contribution of groundwater to lake expansion. The groundwater contribution to the lake includes not only the glacial meltwater that infiltrates the piedmont plain but also other, more isotopically deleted water sources from other basins. It is speculated that the 2003 M_s 6.4 earthquake in the northwest of the Delingha region was a possible mechanism for lake expansion. Earthquakes can enhance crustal permeability and keep fractures open, which promotes groundwater contribution to lakes and in turn causes rapid lake expansion and an increased groundwater level. This study is important for understanding the sources, circulation, and evolution of groundwater in Qaidam Basin.