Abstract: Domestic sewage contains a large amount of organic matter. In the process of sewage treatment, the degradation of organic matter produces a large amount of CO₂, which is an important source of atmospheric CO₂. With the help of dissolved inorganic carbon carbon isotope composition (δ¹³C-DIC), the degradation mechanism of organic matter in water and the process of releasing CO₂ can be effectively elucidated. However, the changes of δ¹³C-DIC and carbon isotope fractionation in the process of sewage treatment are not clear. A domestic sewage treatment plant was selected as the research object, and sewage samples were collected in different treatment units of the sewage treatment plant. The carbon isotope fractionation mechanism and CO₂ release process were judged by means of dissolved inorganic carbon carbon isotope technology and Rayleigh fractionation model. The results showed that: (1) For the influent of wastewater treatment plant, the concentration of dissolved inorganic carbon (DIC) was the highest in the whole treatment process, which was (7.62±0.16) mmol/L, and partly from the dissolution of carbonate rocks. The saturation index of calcite was the largest (0.404) and tended to precipitate, and the calcite tended to dissolve with the wastewater treatment process. (2) The effluent δ¹³C-DIC (mean value −10.45‰±0.28‰) was the highest, and the influent δ¹³C-DIC (mean value −12.40‰±1.07‰) was not the lowest, but higher than that in the biological treatment area (anaerobic, anoxic and aerobic tank). It was due to the degradation of organic matter and the dissolution of calcite to produce and release CO₂, resulting in a decrease in the DIC concentration of sewage in the biological treatment area, an increase in the proportion of dissolved CO₂ from the degradation of organic matter in sewage DIC, and a significant decrease in sewage δ¹³C-DIC. (3) From the beginning of the aerobic zone, the organic matter was rapidly consumed under the action of aerobic bacteria, a large amount of CO₂ escaped, and the DIC concentration was significantly reduced, resulting in an increase in δ¹³C-DIC in the water body and accompanied by a strong isotope fractionation process. The carbon isotope fractionation coefficient was calculated to be 0.992 7. The research results can provide support for optimizing the carbon emission accounting of urban domestic sewage plants.