Abstract: A method for determination of free phytosterols in tobacco was developed with dichloromethane extraction, micropore membrane filtration and gas chromatography/mass spectroscopy/selected ion monitor.The free phytosterols in tobacco samples, mouldy tobacco, and cigarette samples were tested. The results showed that:1) the recovery of free phytosterols was from 93% to 103%, the detection limit was 5.12 to 8.43ng/g and RSD below 6%;2) the oriental tobacco samples had the highest ergosterol level, followed by Virginia tobacco, and burley tobacco the lowest;3) the free phytosterols in cigarette filler significantly correlated with the benzoa pyrene delivery in mainstream cigarette smoke.The conclusion was that the method was suitable to quantitative analysis of free phytosterols in batches of tobacco samples.