The Chadianqiao underground river is located in karst areas in southwestern China, where the movement of the water, including rainwater, surface water, and ground water is extremely rapid. The groundwater in the area is an important source of local drinking water. This study measured the sulfate (SO42-) concentration of rainwater, surface water, and groundwater in the drainage basin. Deuterium (DH2O) and oxygen (18OH2O) isotopes were used to track the source of the surface water and groundwater. Sulfur (34SSO4) and oxygen (18OSO4) isotopes in the SO42- were employed to identify the source of SO42- in the surface water and groundwater, and, additionally, to quantify the proportion contributed by the different SO42- sources at the underground river outlet. The results demonstrated that 1) the major source of surface water and groundwater was precipitation. The SO42- was active in the dissolution of carbonate rocks in the river basin together with HCO3-, as well as in the dissolution of the calcium/magnesium particles in the rainwater. 2) The SO42- concentrations in the different water bodies were (in descending order) surface water > groundwater > rainwater. The SO42- was more enriched in the Chadianqiao underground river basin than in the adjacent river basins. 3) The 34SSO4 and 18OSO4 values of the surface water ranged between -12.98‰ and -10.19‰, and -0.54‰ and +9.13‰, respectively. The 34SSO4 and 18OSO4 values of the groundwater ranged between -14.32‰ and +16.58‰, and +2.81‰ and +14.35‰, respectively. The SO42- in SW02 mainly derived from precipitation, whereas that in SW01, SW03, GW02, GW03, and GW06 mainly derived from coal. The SO42- in GW05 derived from gypsum, whereas that in GW01 and GW04 derived from mixed sources. 4) At the underground river outlet, 13% of the SO42- derived from precipitation, 40% from sulphide oxidation in coal seams, and 47% from the dissolution of gypsum.