Abstract: In order to explore the effect of different preparation methods on the quality of tobacco seed oil, tobacco seed oil was produced by two methods of cold pressing and hot pressing. Yield, physical and chemical indexes, fatty acid compositions and contents of main lipid accompaniments of oil produced by the two methods were compared and analyzed. The results showed that oil yield of hot-pressed oil was higher and the color of the oil was darker. Acid value and peroxide value were significantly higher than those of cold-pressed oil. Unsaponifiable and iodine values were slightly higher than those of cold-pressed oil. There was no difference in fatty acid compositions and content between hot-pressed and cold-pressed oils. Besides, the contents of phytosterols, squalene and vitamins in cold-pressed oil were significantly higher than those in hot-pressed oil. The results of this study provide some reference for further development and utilization of tobacco seed oil in terms of its properties and compositions.