Abstract: In order to quickly and accurately determine the content of alkaloids in tobacco leaves, the extraction conditions were optimized, and a method for the determination of nicotine, anatabine, anabasine and nornicotine contents in tobacco by HPLC was established. The optimized detection steps are as follows: 100 mg/20 mL solid-liquid ratio, 5% methanol was extracted by oscillation at 25 ℃ for 30 min, the chromatographic conditions were C18 column (250 mm×4.6 mm, 5 μm), mobile phase A (90% water +10% methanol, pH 6.8, 0.15% sodium acetate), mobile phase B (90% methanol +10% water, pH 3.8). Nicotine was in the range of 0.1-50 μg/mL and the other three alkaloids were in the range of 0.005-2 μg/mL, with the standard curves of the four alkaloids all havig good linear relationship (R² > 0.99). The limits of detection (LOD) of nicotine were 0.000 65 μg/mL, and the average recoveries were 100.79%, RSD 3.11%; the LOD of anatabine were 0.088 4 μg/mL, and the average recoveries were 98.53%, RSD 3.75%; the LOD of anabasine were 0.034 8 μg/mL, and the average recoveries were 98.70%, RSD3.63%; the LOD of nornicotine were 0.150 9 μg/mL, and the average recoveries were 101.33%, RSD 4.61%. A total of 11 different samples of 4 different types of tobacco leaves were tested, and the results were reliable. The method is simple and fast, sensitive and repeatable, and can meet the requirements of detecting four alkaloids in tobacco samples.