The invention discloses a detection method of polyamine substances in tobacco roots, stems and leaves. The method comprises the steps that vortex extraction is conducted on tobaccos to obtain polyamines through a pre-cooled perchloric acid solution, derivatization is conducted through 3,5-dinitro benzoyl chloride, extraction and concentration are conducted on a derivative product through vortex auxiliary-liquid-liquid microextraction, and detection is conducted through an ultra-efficient liquid chromatography-photodiode array detector. In addition, in order to obtain the best enrichment factors and the recovery rate in vortex auxiliary-liquid-liquid microextraction, method optimization is conducted through fractional factorial design and Doehlert design. Compared with other methods of analyzing and detecting the polyamines, the detection method of the polyamine substances in the tobacco roots, stems and leaves has the advantages of being rapid in derivative reaction, stable in product, high in sensitivity, good in reproducibility, green and friendly to environment and the like, and accurate qualitativeness and quantification can be conducted on the polyamines in the tobacco roots, stems and leaves. In addition, other four kinds of polyamines, namely, tyramine, 1,3-propylene diamine, high spermidine and canavalmine are identified through high-resolution mass spectrometry, wherein the canavalmine is found in the tobaccos for the first time.