Abstract: Aminopeptidase is one kind of exopeptidase which can hydrolyze the hydrophobic amino acid one by one in the amino terminus of the peptide chain. It has many potential applications in various fields. Optimization of culture medium for aminopeptidase fermentation of Aspergillus oryzae was carried out using one-factor-at-a-time method. The optimized medium compositions were as follows: 2% soybean powder,0. 3% K2HPO4,0.2% oleic acid and pH5. 0. By liquid culture in above mentioned medium at 30℃,200 r /min for 48h,the activity of aminopeptidase was up to 497. 13 U /mL and increased by more than 3 times compared with that of using initial medium. This Aspergillus oryzae could produce a high yield of aminopeptidase quickly in low-nutrient conditions,so it was a good candidate for the production research of aminopeptidase.