The content of deoxynivalenol (DON) in whole wheat flour was detected by high performance liquid chromatography (HPLC). The influence of pretreatment method, extraction mode and extraction time, standard curve and chromatographic column on the determination were analyzed. The results indicated that in contrast to the multifunctional purification column treatment method, the immuno-affinity column method used 0.1% of tween leachate sample solution for ageing flour with deep color, the sample was treated clean and the measured chromatogram had less peaks, which was beneficial to the separation of the target peak. Compared with the chromatographic conditions of GB 5009.111-2016, the chromatographic column of C18 and 5 m 4.6  250 mm was more suitable for the determination of the content of vomiting toxin in whole wheat flour.