Abstract: As a marker for isotope tracing technology, carbon-13 is widely used and the market demand is on the rise. In order to explore the separation method of carbon-13 isotope, a gas diffusion separation experiment using ethanol as the processing gas was carried out, and a cascade calculation was carried out on the basis of the single-stage experiment. The results of the single-stage separation experiment show that the overall separation factor for unit mass difference of ethanol separation by gas diffusion can reach 1.008 9 under the existing experimental conditions. It is feasible to use ethanol as processing gas to separate carbon isotopes by gas diffusion method. Through the cascade calculation, it can be known that using natural ethanol as the feed, combined with the feasibility and economy of separation, after a square cascade or matched abundance ratio cascade separation, a heavy fraction with the carbon-13 isotope abundance greater than 25% can be obtained. If ethanol with a carbon-13 isotope abundance greater than 25% can be converted into a suitable form of carbon compound, it can be further separated to obtain a higher abundance of carbon-13 isotope.