Alpha-glucosidase inhibitor is one of the six groups of medicine used for treatment of type 2 diabetes mellitus. In last decade, Aspergillus oryzae has been demonstrated to be able to synthesize alpha-glucosidase inhibitor under suitable conditions. In this study, 35 strains of mold had been isolated from 67 studied samples of koji mold, maize, rice, soybean and Thuoc Bac yeast, of which the strain AOT10 had the ability to inhibite approximately 35 percent alpha-glucosidase substrate in Czapeck- dox medium containing 1 percent soybean. The selected strain was identificated as species of Aspergillus oryzae by two methods including morphological characteristics and DNA amplifiation. The isolated mold was identified Aspergillus oryzae by using biochemical analysis for morphological characteristics and DNA method showed the ITS1-5.85-ITS2 region, typically for Aspergillus oryzae, and its nucleotide sequence with 99 percent of identical as the standard sequence of Aspergillus oryzae published in the International Gene Database Bank. The strain A. oryzae AOT10 produced the highest amount of the inhibitor that inhibited 75.3 percent of alpha-glucosidase at 30"C and 55 percent of humidity soybean for 48 hours of cultivation.