Groundwater is usually downstream of the soil layer, which is one of the most important carbon reservoirs in the terrestrial ecosystems. Assessing the source, migration and transformation processes of carbon in water cycling system is of importance to accurately estimate groundwater carbon sink, which may has significant implications for the study of regional carbon balance. In this study, 474 groundwater samples were collected in Tarim Basin, including 461 groundwater samples from the circular oasis area around the basin, 3 samples as a set of stratified groundwater data in desert areas and 10 samples of different rivers. Samples were analyzed to determine the source, migration and transformation of carbon in groundwater system of Tarim Basin. It showed that the carbon in groundwater mainly originates from the water infiltration through soil layer in the vadose zone rather than the dissolution of carbonate minerals in the aquifer system, revealing that soil carbon pool is the primary source of carbon elements in groundwater. This is the key to determining whether groundwater carbon cycling becomes a carbon sink. Meanwhile, the mineral dissolution during the process of groundwater runoff is the main source of other major elements in groundwater, for example, chlorine, sulfur, potassium, sodium, calcium and magnesium. Moreover, the migration and transformation process of carbon in the aquifer systems is vital to determine the hydrochemical variation and controlling mechanism of carbon sink in groundwater. As the total dissolved solid (TDS) in groundwater increases during the runoff process, bicarbonate may be converted into carbonates and free carbon dioxide under the water-rock interactions, and thus more carbon are trapped in deep aquifers, which can increase the total carbon sink amount in groundwater. At the same time, this process will reduce the carbon content in the groundwater samples with high total dissolved solids, which needs to be taken into account in the evaluation methods. Finally, a new estimation method of groundwater carbon sink was established by considering the amount of groundwater circulation and carbon contents in different drainage areas. The method was used to estimate the groundwater carbon sink in the study areas. The results indicate that the groundwater carbon sink of the seven main drainage areas in the Tarim Basin is about 1.51 million tons per year (counted by carbon). The case study suggested that the groundwater carbon sink is one of the most important subsequent components of soil carbon pool, which may strongly influences the terrestrial carbon sink system and is crucial for global carbon balance.