Abstract: “Tobacco-free” or synthetic nicotine products have been favored by the majority of young people, which undoubtedly increase the potential health risks and regulatory challenges. Currently, there are few reliable methods for the determination of authenticity of natural nicotine and synthetic nicotine. The analytical techniques based on mass spectrometry have broad application prospects in component sequence analysis, structure analysis, molecular weight determination and component content determination. The synthesis processes of natural nicotine and synthetic nicotine are different, and their isotopic fractionations are also different. Here, a method of tandem mass spectrometry based on isotope model peak was developed using this feature. The source or naturalness of nicotine is verified by comparing the abundance and area ratio of M+H⁺ and M+H+1⁺ peaks of nicotine, and the abundance ratio of M+H⁺ and M+H+1⁺ peaks as fragments of parent ions. A total of 47 experimental results showed that the m/z 164.1, 163.1 peak abundance ratios and the area ratios of natural nicotine and synthetic nicotine in MS spectra were significantly different. In addition, in MS/MS spectra, the abundance ratios of fragment ions of m/z 163.1, 164.1 also had significant differences. Among the variables with significant differences, the overall abundance ratio of synthetic nicotine was higher than that of natural nicotine. The evaluation model for the identification of natural nicotine and synthetic nicotine was further constructed by combining chemometric methods. Using OPLS-DA modeling, the discriminant accuracy was 97.87%. Moreover, rankings of importance of variables differed depending on the model of the evaluation. 164.1-85.1/84.1, 164.1-85.1/(84.1+85.1), 163.1-131.1/131.1, 163.1-131.1/(130.1+131.1) were the key discriminant variables. This method has the advantages of simple operation, rapid operation and no need for quantitative analysis, which will help to accurately identify, label and supervise tobacco products from different nicotine sources on the market.