Abstract: In order to investigate the effects of moisture content, tobacco powder particle size and pH on the nicotine release rate in snus, an analytical instrument to simulate the nicotine release in buccal cavity was developed with an accompanied method for the in vitro release of nicotine. The snus samples were prepared according to the Swedish Match GothiaTek^® standard. The in vitro release behaviors of nicotine were measured under different moisture contents, tobacco particle sizes and pH values. The results showed that:1) Comparing with the sample at 10% moisture content, the nicotine release rate at 30% moisture content was raised by 40% within 2 min. When the moisture content was above 30%, nicotine release rate did not change significantly. 2) Within the initial 2 min, the nicotine release rate for samples with 180 μm tobacco particle size was three times that of 1 700 μm. 3) The nicotine in the samples could be effectively released at pH 6.7, 7.4 and 8.5. 4) The 3-factor models of nicotine release for the snus samples followed an exponential function with the fitting coefficient R² higher than 0.95. The results of this study provide a theoretical and practical reference for the product quality and nicotine release for snus development.