A structure identification method of flavonoids in tobacco was developed based on liquid chromatography mass spectrometry (LCMS). The two-step precursor ion scan method was used to identify flavone aglycones and glycosidic conjugates. The substitutional position of glycosidic conjugates was deduced by retention rules in high performance liquid chromatography. The identification was confirmed by MS² spectrum and authentic standards. With this method, 18 flavonoids were identified from tobacco leaf and flowers and 10 of them were identified from tobacco for the first time. The extraction solvent and mobile phase of liquid chromatography method were optimised. The best extraction result can be obtained by using water-acetonitrile solvent (1:3, v: v) and the best separation result can be obtained by using both formic acid and ammonium acetate as additive agent. The method was then applied in the determination of flavonoids in different types of tobacco and the correlationship between flavonoids and flavour related compounds in flue-cured tobacco. The result showed that Turkish tobacco has the highest flavonoid content while Burley has the lowest content. The content of dihydroquercetin has a significant positive correlationship with flavour related compounds such as maillard reaction product, phenylalanine degradation product and carotenoid degradation product, which prove the opinion that flavonoids are the precursors of flavour related compounds.