Abstract: Melamine analysis detection method has become one of the leading subjects of interest to experts in the field of chemical, food and the environment. The use of quantum chemistry and molecular mechanics, such as a computational chemistry research fragmentation mechanism, play an irreplaceable role in the experiment. According to the related reported experimental phenomenon, the first-principles calculation based on density functional theory was used considering spin polarization effect of atoms. The stable geometry of each mass fragment ion’s excimer was optimized for each fragment ions, and the fracture energy of bond formation was calculated at the level of B3LYP/6-311 G (2d,2p). Mulliken atomic charge distribution of three kinds of intermediate ion fragments was also analyzed, thus fragmentation pathway of melamine was obtained. The calculated results show that pseudo-molecular ions m/z 127 of melamine are cleaved by ring fracture mode, and a large number of fragment ions m/z 85 and m/z 68 are generated mainly by way of stepwise cracking. Subsequently, some fragment ions m/z 68 are further cleaved to form fragment ions m/z 43. Using positive ion multi-reactions monitoring (MRM) technology of triple-quadrupole mass spectrometer, major fragment ions of the melamine C₂H₅N₄⁺ (m/z 85), C₂H₂N₃⁺ (m/z 68) and CH₃N₂⁺ (m/z 43) were detected, and their signal intensity of mass spectrometry were in turn decreased in our experimental observations, which further verify the correctness of the theoretical calculation of the dissociation energy. In addition, the theoretical calculation also found that the molecular mass spectrometry of excimer ion was mainly influenced by the Mulliken charge distribution of the fragment ions and the chemical bonds between atoms. This method can be used to identify the melamine structure and provide a theoretical basis for the detection, research and application of melamine.