Abstract: 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 can enhance the sample signal strength and ensure the optimal internal precision of the sample test results. Acidifying the water samples to pH＜3 Phosphoric acid can eliminate the influence of HCO₃ ⁻ in karst waters. A nitrogen-blowing apparatus at a 250mL/min flow rate for 5-10 minutes can remove 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 deviation 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 is 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.