The long-term preservation of Aspergillus oryzae was achieved through the use of brown rice as the main culture medium and vacuum drying (-0.1 MPa) and cryopreservation (-80 ℃). Koji prepared using A. oryzae that had been preserved for 20 years by this novel method exhibited neutral protease, acidic protease, amylase, and glucoamylase activities of 1871, 319, 256, and 2110 U/g dry Koji, respectively. In soy sauce prepared using the aforementioned Koji, the contents of total nitrogen, formaldehyde nitrogen, total sugars, reducing sugars, and salt-free solids and the rate of utilization of proteins in the raw materials were 1.53, 0.92, 5.39, 3.52, and 19.88 g/100 mL and 82.20%, respectively. These indicator values were significantly superior to those of Koji and soy sauce prepared using A. oryzae preserved for 20 years by the conventional tube freeze-drying method (p<0.05) and were not significantly different from those of Koji and soy sauce prepared using A. oryzae preserved for 5 years by the conventional tube freeze-drying (p>0.05). According to the hierarchical cluster analysis results, compared with the conventional tube freeze-drying method, the novel method prolonged the preservation of A. oryzae by 15 years, reduced the number of times needed for its rejuvenation during actual production, and lowered the possibility of its mutation and contamination during passage. These attributes would enhance the quality stability of soy sauce.