The influx of a large amount of sulfate (SO42-) into water bodies can deteriorate the water environment quality. During the period from 2019 to 2023, water samples from the Pingzhai Reservoir Basin in the southwestern karst area, precipitation samples, and sewage samples were collected. The physical and chemical indicators of the water bodies, as well as the characteristics of sulfate sulfur and oxygen isotopes (δ34SSO4, δ18OSO4), hydrogen and oxygen isotopes of water (δDH2O, δ18OH2O), and dissolved inorganic carbon isotopes (δ13CDIC) were analyzed during the monitoring period. Qualitative and quantitative studies on the sources of SO42- in the water bodies were carried out. The hydrochemical types of the basin are mainly of the Ca-HCO3 type and Ca-HCO3-SO4 type, and the hydrochemical characteristics are influenced by the dissolution of carbonate rocks. Isotope characteristics reveal that the biogeochemical process of SO42- in the Pingzhai Reservoir Basin is dominated by oxidation (when sulfides are oxidized, on average, 61% of the oxygen comes from the surrounding water bodies). During the monitoring period, the sources of SO42- in the basin are mainly sulfide oxidation, soil organic sulfur, and agricultural sulfur fertilizers. The results of quantitative calculations show that sulfide oxidation has the highest average contribution rate (72.9%), followed by soil organic sulfur (14.1%) and agricultural sulfur fertilizers (12.9%). The results of uncertainty analysis indicate that the contribution rate of agricultural sulfur fertilizers is the most stable, while that of sulfide oxidation shows relatively large uncertainty. The research results can provide data reference and scientific basis for the protection of water environment quality in the Pingzhai Reservoir and similar karst areas.