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Carbon sequestration resulting from carbonate rock weathering is closely linked to the global carbon cycle and has turned out to be important in the adjustment of atmospheric CO2 levels. Traditional karst dynamic models based on water–rock–gas interactions underestimate carbon sink fluxes related to carbonate rock weathering because they ignore the utilization of dissolved inorganic carbon (DIC) by aquatic organisms. In this study, a new model based on water–rock–gas–organism interactions was applied in the Pearl River basin, China, to recalculate atmospheric CO2 consumption and to develop an accurate estimation model for carbon sink fluxes at catchment scale. Stable carbon isotope (δ13C) and C/N ratios were used in the counting processes. Data were collected from published literature as well as through field investigation and laboratory analysis. Results show that the Pearl River carbon sink in the Pearl River is 4.31 × 109 kg/a, i.e., 15.8 × 109 kg of atmospheric CO2 per year. Of this, the carbon sink resulting from carbonate rock weathering amounts to 2.14 × 109 kg/a, i.e., 49.7 % of the total. The three largest tributaries of Pearl River, Dongjiang, Beijiang, and Xijiang, respectively absorb 0.5 × 109, 1.19 × 109, and 2.62 × 109 kg of carbon from the atmosphere annually, accounting for 12, 28 and 60 % of the river’s total carbon sink. When compared with the results of previous researches that disregarded the role of aquatic organisms, the new calculation method provides a carbon sink flux value that is 1.2–3.3 times higher, and 1.6 times higher on an average. To improve the calculation accuracy of atmospheric CO2 consumption in global karstic rivers, further research is needed regarding carbon sequestration mechanisms that involve aquatic organisms.