Abstract: A method for the extraction and preparation of polyphenol reference substances from peduncle of tobacco by accelerated solvent extraction and pre-HPLC was proposed. The sample was crushed and extracted with ethanol (80+20) solution under static condition at 110 ℃ for 10 min, and the extract was swept for 60 s by N2-blowing and purified by MCI resin and Sephadex LH 20 gel. The eluate collected was taken up with acetone and used for preliminary separation by pre-HPLC, and for further separation by semi-Pre-HPLC, and five polyphenols were obtained, which were identified by NMR to be rutin, chlorogenic acid, quercetin, naringenin and methyl caffeate. The purity of the 5 polyphenols were more than 99%, which could be used as reference materials.