Soil organic carbon (SOC) on the Qinghai-Tibet plateau is of global significance, due to the high carbon density and potential positive feedbacks to climate warming. Qinghai Lake watershed is an important soil pool in Qinghai-Tibet plateau, but the magnitude and characteristics of SOC stock in the high-altitude basin were not clear. To understand better how much SOC was stored and its spatial patterns in the watershed, this study analyzed the vertical variation of SOC content, quantified its influence by varied environmental factors, and further predict its storage in the entire basin. Results showed that, the SOC content decreased with the increasing depth, and was the highest in the alpine meadow in all depths, following by the alpine steppe and temperate steppe, respectively. It was generally higher in the permafrost region than that in the non-permafrost region at all depths. Soil profile depth, soil water content and NDVI could together explain about 72.6% of total variation in SOC density. The regression-kriging method was the best upscaling approach for the prediction of SOC in the study area. Approximately 317 Tg organic carbon was stored in the entire soil profile of Qinghai Lake watershed, in which 136, 140, and 41 Tg were at 0–30 cm, 30–100 cm, and below 100 cm, respectively. The higher SOC density mainly appeared on the northeast and southern region of Qinghai Lake, while the high-altitude area in the northwest of the watershed had the lower values. Moreover, it increased firstly and then decreased with the increase of altitude, with the maximum values at 3350–3400 m, which was dominated by the elevational variation of soil profile depth and NDVI. Overall, the comprehensive assessment of SOC demonstrated high storage and strong heterogenicity of soil carbon in the Qinghai Lake watershed, and it is critical for the sustainability of regional terrestrial ecosystem with future climate change.