Abstract: With the promotion and popularization of carbon-13 breath detection test, the market demand for carbon-13 isotope products is on the rise. In order to explore the industrial separation method of carbon-13 isotope, a gas diffusion separation experimental research using carbon dioxide as processing gas to separate carbon isotopes was carried out. According to the average free path of carbon dioxide, an organic polymer porous membrane with an average pore diameter of 55-65 nm was used as the separation membrane, and a single-stage separation device was developed. Through the single-stage diffusion separation experiment, the influence of the number of separation membrane layers and pre-membrane pressure on the membrane diffusion separation performance was studied. The results show that the gas diffusion separation device using organic polymer porous membrane has a separation effect on carbon dioxide, and the maximum concentration separation coefficient of organic polymer membrane diffusion separating carbon dioxide was 1.004 9 under existing experimental conditions. On the basis of single-stage diffusion separation experiment, combined with the compressor and cascade design, multi-stage diffusion separation experiment will be further carried out to explore the application prospects of carbon-13 isotope separation by gas diffusion method.