Abstract: In order to established the method of determining total solanesol in tobacco efficiently and accurately, we studied the conditions of simultaneously extracting and saponifying solanesol from tobacco, including solvent, temperature, liquid ratio, saponification dosage, extraction time and ultrasound-assisted instruments conditions of ultra-high performance liquid chromatography. Hexane was used as solvent for extraction with liquid-material ratio of 50:1 and 0.1 moL/L sodium hydroxide in ethanol solution was used as saponification agent. The ultrasonic frequency used was 45 kHz, temperature of extraction and saponification was 40 ℃, and time of extraction was 30 min. Under this condition, extracting and saponifying solanesol from tobacco were finished simultaneously, and saponification agent separation was done in the same centrifuge tube. The separation of target compound was performed on an ACQUITY UPLC BEH C18 column using methanol-acetonitrile (50:50) as mobile phase by gradient elution. Flow rate was set at 0.5 mL/min, column temperature was 35 ℃ and injection volume was 5 μL. The quantitative wavelength of UV detector was set at 208 nm. The results indicated that the calibration curve was linear in the range o f 0.67-84.1 mg/L, the limit of detection (LOD, S/N=3) was 0.07 mg/L, and the limit of quantification (LOQ, S/N=10) was 0.012%. The standard addition recoveries of blanks and samples were 97.9%-104.7% and 93.4%-100.0%, respectively, with relative standard deviations (RSDs) of 1.34%-2.43%. The method was simple, rapid, precisive, accurate, and solvent saving, suitable for batch testing .