Using density functional theory(DFT) B3LYP method,the preferred molecular conformations and the optimal configurations of kaempferol,quercetin and myricetin quasi-molecular ions in electrospray ion trap mass spectrometry(ESI-ITMS) negative ion mode were studied by the full geometry optimization and the relaxed dihedral potential energy scan.The optimal configurations of three flavonol quasi-molecular ions were confirmed in the aspects of energy parameters,configuration parameters,and mass spectrometry experiments.The results indicated that the degrees of dihedral D(1,2,1′,6′) for kaempferol,quercetin and myricetin were close to 0°,and the preferred conformations of three flavonol compounds were respectively coplanar including A,C and B ring.In the negative ion mode,the complexities of MS2 spectra which were determined by the automatic and manual methods for kaempferol,quercetin and myricetin were successively weakened.The optimal configurations of quasi-molecular ions were the structures lost the hydrogen of phenolic hydroxyl,with the conjugated chain increasing and the conjugated effect strengthening.Two quasi-molecular ion configurations for kaempferol existed,that were loss of 4′-OH hydrogen and 7-OH hydrogen,and the total energy of loss of 4′-OH hydrogen configurations for quercetin and myricetin were the lowest and the most stable.The stabilities of quasi-molecular ion configurations for kaempferol,quercetin and myricetin were increased successively.The analysis of the study can be used as a reference for further exploration of the fragmentation pathway and mechanism of flavonol compounds in ESI-ITMS negative ion mode.