Several polyphenolic compounds which were derived from the fermented soybean had been identified and investigated for their potential effects on some diseases. However, such a biotransformation phenomenon is seldom discussed and has not been studied by using submerged culture with isoflavone, such as pure daidzein, as a substrate. In this study, the conditions for hydroxylation of pure daidzein into 8-hydroxydaidzein by Aspergillus oryzae were systemically investigated. Our results indicated that the optimal culture conditions for the hydroxylation were 200 rpm and pH 6. Moreover, the production was significantly increased 57.4% by an optimized concentration of 3 g/L MgSO₄. On the other hand, 77.8% high biotransformation efficiency of 8-hydroxydaidzein could be obtained by providing pure daidzein; that can avoid many derived polyphenolic compounds were generated by isoflavone biotransformation. Our results suggest that the biotransformation of 8-hydroxydaidzein can be performed through a highly selected catalysis under specified conditions using certain microorganisms.