For the fast and accurate determination of sugars in tobacco leaf, a method for the determination of sugars (fructose, glucose, sucrose, maltose) in tobacco leaf by an ultra-high performance liquid chromatography (UPLC) was established，taking a novel extraction vial as the pretreatment device. The results showed that: 1) the novel pretreatment device could make the extraction, filter and transfer as one step, make the procedure of the pretreatment more efficient. 2) The separation of the sample was operated on ACQUITY UPLC BEH Amide column (2.1 mm×100 mm, 1.7 μm), using 75% (v/v) acetonitrile containing 0.2% triethyla-mine as the mobile phase at a flow-rate of 0.2 mL/min. The 4 analytes could be separated smoothly with good linearity in the range of 5～250 μg/mL, with the correlation coefficients larger than 0.999. The limits of detection (LODs) were further evaluated (1.0～3.2 μg/mL). Under three different spiked level The mean recovery of the target compounds was 91.0%～102% for 4 sugars and the intra-day and inter-day precision were below 5%. 3) Different contents of sugars in different type tobacco leaf were found with the established method. The new method, with the novel pretreatment vial for extraction and the HPLC for separation, can be used for analysis of sugars in tobacco samples.