Abstract: In order to determine the optimal experimental parameters for the extraction of petroleum ether extract from tobacco with fully-automatic Soxhlet extractor, the single factor tests and response surface methodology (RSM) were executed to optimize the extraction parameters. The flue-cured tobacco standard material of petroleum ether extract (GBW08514a) was used. The experimental results showed that the optimal extraction parameters were as follows: boiling time/leaching time: 15/90 min, extraction temperature: 100 ℃, extraction solvent volume: 85 mL. Under the optimal parameters, the content of petroleum ether extract of the flue-cured tobacco standard material was 4.70% (RSD/%=1.85). Consequently the optimum parameters with fully-automatic Soxhlet extractor for the extraction of petroleum ether extract from tobacco were established by response surface methodology. The discrepancy of the results applied with the proposed method and the traditional method was negligible. Moreover this method saved the labor time and greatly improved working efficiency, which was more suitable for the rapid determination of the petroleum ether extract for a multitude of tobacco samples.