The invention relates to detection of flavonoids in tobacco, in particular to a metabonomics method for detecting multiple flavonoids in fresh tobacco leaves.The method is characterized in that flavonoids in the tobacco leaves are extracted through an ultrasonic extraction method, and rutin, quercetin, naringenin, kaempferol glucoside, astragalin, naringin and isoliquiritigenin in the fresh tobacco leaves are detected through UPLC-QqQ-MS/MS.Compared with the prior art, the method has the advantages that accuracy and sensitivity are high, repeatability is good, and the method is suitable for simultaneously and quantitatively detecting the seven flavonoid compounds in plants and can be used for research on metabolic pathways and gene functions of plant flavonoid matter.