Abstract: A high performance liquid chromatography with fluorescence detection for determining the benzo-apyrene in mainstream cigarette smoke was developed. The extraction, clean up with Florisil and chromatographic conditions were tested. The Kentucky reference cigarette 3R4F and domestic Virginia type reference cigarette samples and 50 domestic cigarette brands samples were parallel tested with the developed method and national standard method. The results showed that:1)tobacco samples were extracted more effectively with cyclohexane under ultrasonic for 40 minutes;2)benzoapyrene was separated effectively in 30 minutes by reversed-phase HPLC with an Agilent ZORBAX Eclipse PAH column under a gradient elution with acetonitrile and water as mobile phase at a flow-rate of 2.0 mL/minute;3)the recoveries of benzoapyrene ranged from 98.13% to 101.36% with the relative standard deviation (RSD)of 1.29% to 4.32%;4)there was no significant difference between the results determined with the developed method and national standard method. This method is suitable for determining benzoapyrene in mainstream cigarette smoke.