Abstract: A stable isotope ¹³C separation three-stage cascade equipment using carbon monoxide (CO) cryogenic rectification was designed. The distillation columns were packed with self-developed high efficiency structure packing PACK ¹³C of 38 m height. The columns diameters are 0.15 m, 0.08 m and 0.05 m respectively. The uniform design method was taken to optimize the cascades equipment. The effects of the reflux ratio, feed amount, inter-columns material flow on the ¹³C product purity and the energy consumption were analyzed synthetically. The mathematical model of the product purity relative to the reflux ratio, feed amount, and inter-columns material flow and the mathematical model of the energy consumption relative to above decision variables were obtained by quadratic polynomial stepwise regression analysis of the experimental data. Then, the optimal operation conditions at the lowest energy consumption were achieved by the method of genetic algorithm. If the ¹³C product purity is more than 93%, the optimal inter-columns material flows are 18.056 mol/h, 236.50 mol/h, 32.400 mol/h and the reflux ratio is 74.824. Compared with the initial designed conditions, the optimal operation conditions can reduce the energy consumption. The method of simulation and optimization could be applied to design ¹³C industrial and realize optimization design in traditional distillation process.