Abstract: To improve the utilization value of tobacco stem extract, molecular distillation was used to separate and purify tobacco stem extract prepared by supercritical fluid extraction. The parameters of molecular distillation were optimized by the response surface method, and the components in the extracted essential oil were determined by GC/MS. The results showed that:1) The highest yield of the essential oil was 10.49% ±0.05%, the optimized processing parameters were distillation temperature 140℃, film scraping speed 246 r/min, and condensing temperature 39℃. 2) Comparing with tobacco stem extract, the contents (mass fraction) of 24 components in the obtained essential oil increased greatly, and the enrichment of eucalyptol was the highest and reached 112 times. 3) The obtained essential oil was used as a flavor for reconstituted tobacco, and the sensory evaluation results indicated positive sensory quality. This method is suitable for purifying tobacco stem extracts, and the obtained essential oil enhances the tobacco-like aroma of reconstituted tobacco products.