Abstract: An engineering research method about “numerical simulation and experiment study” was established in the study of ¹³C isotope separation, at the same time, method of computer aided design was established and engineering experiments were completed in the separation of isotope ¹³C by carbon monoxide cryogenic distillation. Firstly, the basic parameters of ¹³C isotope system were measured by CO cryogenic distillation in a single column; secondly, ¹³C separation process with two-column cascade process was designed by computer aided design, and the optimized parameters of the cascade setup were obtained; finally, the optimized parameters were tested by CO cryogenic distillation experiment in the two-column cascade setup. The research results showed that the engineering research method about “numerical simulation and experiment study” was reliable, which was proven in ¹³C isotope separation; the research method improved the ¹³C isotope separation design level, reduced the test cost, and improved the developing efficiency, which provided a reliable technique for the design of ¹³C isotope industry production plant.