Abstract: The aim was to establish a research method to measure the phenolic and flavonoid which relate to plant lignin biosynthesis. Organic reagent and ultrasonication were used for plant phenolic and flavonoid extract, HPLC was used for the measurement of six plant phenolic and five flavonoid. Tobacco was used as sample to test the HPLC measure method, the result show that HPLC method for the phenolic and flavonoid relate to plant lignin biosynthesis is efficient which can isolate 6 different phenolics and 5 different flavonoids in one test；organic reagent and ultrasonication are useful to extract plant phenolic and flavonoid; the retention time of p-Coumaric, Ferulic, Caffeic, Cinnamic, Sinapic and 4-Methoxybenzoic Acid are 18.11, 20.03, 20.51, 20.76, 22.86 and 26.23; the retention time of Myricetin, quercetin, phloretin, naringenin and kaempferol are 9.06, 12.01, 14..96, 15.23, 16.12. The method made foundation for the research of plant lignin biosynthesis.