Rationale: Flavonoid C-glycosides have a wide range of pharmacological activities. However, there are few mass spectrometric research on C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides. Their fragmentation pattern and differences have not been reported, and the fragment ion library is incomplete. Therefore, it was essential to elucidate the fragmentation patterns of disaccharide flavonoid C-glycosides, which is described in this study. Methods: Four disaccharide flavonoid C-glycosides such as vitexin-4″- O-glucoside were analyzed by ultra performance liquid chromatography-triple quadrupole tandem mass spectrometer (UPLC-MS/MS) in both positive and negative ion modes. each ion and its proposed fragmentation pathways of the four disaccharide flavonoid C-glycosides were analyzed comprehensively. Finally, Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and the established fragmentation patterns have been used to identify disaccharide flavonoid C-glycosides in Odontosoria Chinensis. Results: The fragmentation pathways of C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides. are similar. they both have the mass spectrometric characteristics of O-glycoside and C-glycoside. Fragment ions after mixed pathways of neutral fragments such as saccharide ring fragment, O-glycosides, H 2O and CH 2O detraction appeared in both types of flavonoid C-glycosides, but their relative abundances are significantly different. According to the established fragmentation patterns, di-C,O-saccharide flavonoid glycosides were also found in Odontosoria Chinensis. Conclusion: The fragment ions of m/z 431, 413, 341, 311, 293, 282 in negative ion mode and m/z 293, 282, 577, 559, 541, 523, 529 and 499 in positive ion mode can serves as the main characteristics for identifying C,O-disaccharide flavonoid C-glycosides and di-C,O-saccharide flavonoid C-glycosides.