Abstract: Ethanol is a feasible medium for diffusion separation of carbon isotopes. In order to obtain the separation coefficient of gas diffusion separation of ethanol, the application of elemental analysis isotope ratio mass spectrometry (EA-IRMS) to measure the carbon isotope abundance in ethanol in gas diffusion separation experiment was studied. Through literature investigation, this study optimized the preparation process of ethanol samples, developed the method of EA-IRMS for the determination of carbon isotope abundance of ethanol, tested the stability, and realized the measurement of carbon isotope abundance of ethanol samples. Based on the separation experiment of gas diffusion method, the carbon isotope abundances of refined ethanol and lean ethanol in multiple separation experiments are obtained. The basic total separation coefficient of carbon isotopes by ethanol diffusion can be calculated by formula derivation. This study provides an analytical basis for the future experiment of diffusion separation of carbon isotopes with ethanol as medium.