Abstract: A stable isotope ¹³C separation column was set up by carbon monoxide (CO) cryogenic distillation. Diameter of the column is 45 mm, packing height is 17.5 m, of which enriching section is 15 m and stripping section is 2.5 m. Firstly, computer simulation results were validated by test results. Secondly, tests were replaced by computer simulations in order to obtain the optimal operation conditions in the experimental setup. Comprehensive factors of column pressure, feeding velocity, reflux ratio, withdrawing velocity, and boiling power impacts on the products were studied. Then optimization design of the experimental device was achieved through computer simulations combined with uniform experimental design. The final results show that the optimal operation conditions in the built column are as followings: boiling power, 250 W; column pressure, 54 kPa; reflux ratio, 84. The conclusion is that the method of combination of computer simulation and experimental design could be applied to ¹³C industrial design and could be popularized in traditional distillation process to realize optimization design.