Abstract: Abstract: 1,8-cineole and α-pinene are the main constituents of eucalyptus essential oil and important medicine and chemical raw materials. The primary eucalyptus essential oil was refined by short-range molecular distillation, and the separation characteristics of 1,8-cineole and α-pinene of substances under different temperature and pressure conditions were studied, so as to provide technical method for the fractionation and application of eucalyptus essential oil. Using eucalyptus grandis leaves as material, the primary eucalyptus essential oil was extracted and purified by supercritical CO2 extraction and molecular distillation method respectively. Taking the distillate yield, the content of 1,8-cineole and the content of α-pinene in the distillates, the content of 1,8-cineole and the content of α-pinene in the residues as experimental indices, two-stage molecular distillation purification was carried out. Response surface methodology (RSM) based on a two-variable and five-level center composite rotation design (CCRD) was employed to evaluate the purification effects. Using the free path theory, the effects of different distillation temperatures and distillation pressures on the content of 1,8-cineole and α-pinene in distillates and residues were investigated. The optimization of the process of eucalyptus essential oil refining by molecular distillation was studied. The distillation temperature and distillation pressure had significant influence on the effect of 1,8-cineole and α-pinene fractionation, and the experimental results were optimized by the optimization method of multi response values. Taking distillate as the target product, the optimum conditions were as follows: distillation temperature 38 ℃, distillation pressure 41 Pa, and 1,8-cineole and α-pinene content in distillates of 60.80% and 31.58%, respectively, and the yield of the distillates was 82.06%, which were well matched with the predicated values 61.97%, 33.37% and 86.38% obtained from the models. The smaller error between experimental and predicted values indicated the optimization experiment was successful. The results of variance analysis showed that the regression equations and models were reliable to predict the influences of factors on the distillate yield, and the content of 1,8-cineole and α-pinene in distillates and residues. Molecular distillation purification can effectively purify the eucalyptus oil. The CCRD is suitable for the process optimization of molecular distillation of eucalyptus essential oil. The experimental process did not introduce any organic solvents, and was carried out in low temperature environment, protecting the material composition and characteristics of the structure. The contents of 1,8-cineole and α-pinene were increased by 77.62% and 56.72% respectively after the secondary molecular distillation. Through the investigation of the change of the content of 1,8-cineole and α-pinene with the distillation temperature and the distillation pressure, the effects of distillation temperature and distillation pressure on the content of 1,8-cineole were significantly higher than those of α-pinene. Meanwhile, the effect of distillation pressure on the content of 1,8-cineole oil was higher than that of distillation temperature. In contrast, for α-pinene, the effect of distillation temperature on its content was more significant, and the interaction of temperature and pressure had no significant effect on the content of α-pinene. The result provides a technical reference for the separation and purification of 1, 8-cineole and α-pinene.