The method to determine deoxynivalenol (DON), 3-acetyl- deoxynivalenol (3-A-DON) and 15-acetyl-deoxynivalenol (15-A-DON) in wheat simultaneously by immune affinity column clean-up and high performance liquid chromatography was established. The wheat samples were extracted by ultrapure water, cleared up with the immuno-affinity column (IAC). After drying and constant volume, three toxins, DON、3-A-DON and15-A-DON, were detected simultaneously by high performance liquid chromatograph with UV detector, and compared with that by Liquid chromatography mass spectrometry. The result showed that the detection limit of method was 100 μg/kg for DON, 3-A-DON and 15-A-DON respectively. Recovery rates in wheat spiked with DON, 3-A-DON, and 15-A-DON, ranged from 86.6% to 96.5%. The variation coefficient was less than 10%. This method was suitable for determination of DON, 3-A-DON, and 15-A-DON in wheat, with the advantages of simplicity, rapidness, sensitivity and good selectivity, veracity and precision.