Fusarium graminearum was inoculated on rice medium and cultivated at 28 ℃ for 4 weeks in the dark.The medium was extracted with acetonitrile-water（84∶16），and then first cleaned up with home-made clean-up cartridges to remove pigment and tannin，followed by two steps of semi-preparative liquid chromatography for further purification.The target compound was separated on a Pursuit XRs C18（10 mm×250 mm，5 μm）column using methanol-water（30∶70）as mobile phase，and then purfied with a Semi-preparative SB-CN column（9.4 mm×250 mm，5 μm） using methanol-water（5∶95） as mobile phase.The obtained deoxyuni valenol(DON) was qualified by high performance liquid chromatography tandem mass spectrometry（HPLC-MS/MS） both in positive electrospray ionization mode（ESI+）and negative electrospray ionization mode（ESI-）.The precursor ions of the compound were 319.1［M+Na］+，297.1［M+H］+，355.2［M+CH3COO］-，341.2［M+HCOO］-，295.1［M-H］-，and the major product ions were 175，231，235，249，179，233，237，265，which indicated that the obtained substance was DON.The purity of the prepared DON was determined to be （99.18±0.11）% by the external standard method.Based on the above observations，it was easily concluded that the preparation method for DON was simple，efficient，stable and economical.