Abstract: In order to determine nicotine in tobacco and tobacco products in real time, an online method based on Direct Analysis in Real Time (DART) ionization technique combined with mass spectrometry was established by optimizing parameters such as extraction solvent type, extraction solvent dosage and injection rate. The samples of five commercial cigarettes and five tobacco leaves were analysed by the established method, and the determined values were compared with those obtained using a continuous flow method. The results showed that:1) Under the optimized conditions (30 mL ultrapure water as extraction solvent and sample injection rate of 0.6 mm/s), the analysis could be completed within 20 s. 2) The linear range of the method was 0.01-2.00 mg/mL with R² of 0.999 1, the recovery ranged from 98.5% to 102.3%, and the detection limit and the limit of quantification were 6.05 and 20.17 ng/mL. 3) The relative error between DART-MS/MS method and continuous flow method was 0.24%-2.74%. The established method is rapid, reliable and suitable for the real-time on-line determination of nicotine in tobacco and tobacco products.