Abstract: In order to achieve the stereoselective separation and determination of stereoisomers of dimethomorph in tobacco, a method coupled supercritical fluid chromatography with tandem mass spectrometry (SFC-MS/MS) was developed to quickly separate and determine the stereoisomers of dimethomorph. Tobacco samples were extracted and purified by QuEChERS method, then the dimethomorph stereoisomers were well separated on a Chiralcel OD-3 column with CO₂-methanol as the mobile phase at the flow rate of 2 mL/min. Matrix matching standard working curve was used to conduct quantitative analysis. The results showed that: 1)The stereoisomers of dimethomorph reached baseline separation, and the separation time was only 5 minutes. 2)At the mass fractions of 0.02, 0.20 and 2.00 mg/kg, the recoveries of two isomers were in the range of 87.3%-102.3% with the inter-day relative standard deviations (RSDs) of 3.3%-5.9%. Good coefficients of determination (R²≥0.994 3) were achieved for two isomers over the isomers concentration range of 2-100 ng/mL in tobacco. The limits of detection (LODs) and limits of quantification (LOQs) for two isomers were 0.006-0.008 and 0.021-0.026 mg/kg respectively. 3) Ten tobacco samples were tested by this method, and (E)-dimethomorph (1.18 mg/kg) and (Z)-dimethomorph (1.22 mg/kg) were detected only in one sample. This method is fast, environmentally friendly, stable, reliable, and suitable for the stereoselective determination of dimethomorph isomers in tobacco.