Dissolved inorganic carbon (DIC) holds significant importance within river systems, particularly in karst regions. The concentration of DIC is intricately linked to both biological processes, such as the respiration of organic matter, and geological processes, notably the strength of carbonate weathering. This dual influence exerts a profound impact on regional and global carbon cycles. However, the precise biological and geological origins of DIC in karst rivers remain a subject of debate. In this study, we delineate the sources of DIC in a typical karst catchment (Houzhai catchment) through isotopic analysis coupled with water chemistry examination. The results showed that δ13CDIC and Δ14CDIC variations range from −14.0 ‰ to −7.3 ‰ and − 426 ‰ to −31 ‰, respectively, with average values of −10.5 ‰ and − 180 ‰. Results derived from the isotopic mixing model suggest that approximately 83 % of DIC emanates from the soil CO2, while 17 % originates from carbonate sources. This indicates that biological processes exert a greater influence on DIC within the study area compared to geological processes. Seasonal variation, exemplified by higher DOC concentrations and Δ14CDIC values, alongside with lower DIC concentrations and δ13CDIC values during the wet season indicate increased input from biogenic sources, closely related to temperature and moisture conditions. Our study underscores an unbalanced contribution from biogenic and geogenic sources in karst rivers, highlighting potential inaccuracies in past carbon sink flux calculations. The roles of biogenic and geogenic sources in the carbon cycling of karst rivers, as well as their responses to future climate change, warrant a reevaluation.