Abstract: This test used tobacco as raw materials, methanol as impregnant, extracted nicotine in tobacco by ultrasonic wave, then determined nicotine by gas chromatography. Studied effect of different concentrations of methanol，ultrasonic time and the ratios of material to liquor on quantity of nicotine in tobacco. The results show that extract nicotine in tobacco by Ultrasonic, this method is very easy and quick .When concentration of methanol was 40%, the quantity of nicotine was 41.52 ±0.28μg/g; when ultrasonic time was 45 min, the quantity of nicotine was 39.92±0.25μg/g; when the ratio of material to liquor was 1∶20, the quantity of nicotine was 42.21±0.16 μg/g .And the also studied relationship of different concentrations of methanol（x1）, ultrasonic time（x2）, the ratios of material to liquor（x3） and the quantity of nicotine（y）by using method of multiple regression orthogonal design. Got the regression equation of extraction quantity of nicotine and different conditions : y＝88.936－6.894x1＋2.086x2－2.439x3－3.164x1x3.