8-Hydroxygenistein is one of the orthohydroxygenisteins, which are derivatives of genistein. Although 8-hydroxygenistein is generally produced through the biotransformation of soya isoflavones by using specific microorganisms, the yield of produced 8-hydroxygenistein is low. In this study, we optimized the cultivation medium for 8-hydroxygenistein production. The results of the central composite design indicated that the optimal nutrient concentrations were 35 g/L of glucose, 22 g/L of malt extract, and 4.75 g/L of soya peptone. The maximum product concentration of 8-hydroxygenistein obtained was 77.76 mg/L. Based on literature review, this concentration of 8-hydroxygenistein is the highest yet reported. The effects of operating conditions on the concentration of 8-hydroxygenistein were also determined using a fermenter. After investigating the effects of the agitation speed and aeration rate, a final product concentration of 83.01 mg/L was obtained. Thus, large-scale biotransformation of genistein to 8-hydroxygenistein can be performed using Aspergillus oryzae.