Abstract: In order to further improve the separation effect of 13C isotope by gas diffusion method, on the basis of preliminary experiments, the single-stage diffusion separation parameter optimization experimental study and the preliminary design of the cascade scheme for the production of high-abundance ¹³C isotope are carried out. Under relatively optimized experimental parameters, the overall separation factor of gas diffusion separation of carbon dioxide can reach more than 1.01. Calculate the experimental data of single-stage separation and initially fit the function relationship between the feed flow rate and the pressure before and after the membrane. The multivariate separation theory is used to carry out cascade analysis and calculation for the diffusion separation of carbon dioxide. With natural carbon dioxide as the feed, high abundance ¹³C isotope can be obtained through two cascade separations. The ¹³C isotope abundance of the heavy fraction in the first squared-off cascade separation is greater than 42%, and it is used as the feed for the second squared-off cascade separation, and the ¹³C isotope abundance of the light fraction in the second squared-off cascade separation is greater than 90%.