Abstract: Due to the high content of HCO₃ ⁻ , low content of DOC and complex composition in the karst water, the current wet oxidation method for the determination of δ¹³C_DOC needs to be improved. The study examined the impact of different pure waters, headspace bottle atmospheric equilibrium methods, DOC content, and the removal of bicarbonate (HCO₃ ⁻ ) in water on δ¹³C_DOC values. The experimental results show significant differences in the ⁴⁴CO₂ peak areas produced by different blank pure waters, with the most prominent peak area being 3.8 times the smallest. Using a gas-tight syringe for atmospheric equilibrium enhances the sample signal strength and ensures the optimal internal precision of the sample test results. Acidifying the water samples to pH<3 with phosphoric acid eliminates the influence of HCO₃ ⁻ in karst waters. A nitrogen-blowing apparatus at a 250mL/min flow rate for 5−10min removes the free CO₂ present after acidification. Under the above experimental conditions, the deviation between the test results and the calibrated reference values for karst water samples with DOC content greater than 2mg/L is better than 0.40‰ after blank correction. The difference in the measured results of δ¹³C_DOC in karst water samples measured by the wet oxidation method compared to the total organic carbon analyzer-stable isotope mass spectrometer online method was less than 0.30‰. The BRIEF REPORT is available for this paper at http://www.ykcs.ac.cn/en/article/doi/10.15898/j.ykcs.202405130108.