Cereal and legume kernels have many α-amylase inhibitors that they have potential to inhibit the α-amylase activity from variant sources. The subject of this study was extraction of wheat seeds, partial purification of α-amylase inhibitors and the assessment of their inhibitory activity upon α-amylase of Aspergillus oryzae. In this regard, we precipitated extractions as three parts with different saturation percentages of ammonium sulfate. Then, we dialyzed, in order to remove ammonium sulfate and better check of their inhibitory activity upon A. oryzae α-amylase. With the assessment of the inhibitory activity of each sample on the basis of their absorbance in 540 nm and in comparison to the absorbance of negative control, we saw that only the fraction of 30% to 60% (F30-60%) of wheat extract had inhibitory activity upon defined α-amylase. Inhibitory percentage of this fraction was 95.77. In addition, for more characterization of these inhibitors, we conducted SDS-PAGE analyses. By assaying the activity of these inhibitors upon other α-amylase such as salivary and pancreatic α-amylases, this may enable us to use them as α-glucosidase inhibitors such as Acarbose and Miglitol for the treatment of type II diabetes. Also, the results might be applied in agriculture to produce transgenic plants that express α-amylase inhibitors against α-amylase of pests.