This study was performed to verify the possibility of manufacturing Rhizopus oligosporus 'Koji', which is cultured using a range of different substrates. The growth characteristics of R. oligosporus were therefore investigated for the preparation of R. oligosporus 'Koji', and the optimal incubation conditions were determined to be 30 ℃ for 24 h, with an optimal NaCl concentration were 5%. Based on these results, R. oligosporus was incubated using three substrates, namely beans, rice and a mixture of beans and rice, for 6 d, and the enzyme activities and volatile compounds of the fermentation products were investigated. On the fourth day of incubation, the α-amylase activities of the rice and the mixed substrates were 155 and 42 unit/mL, respectively, while on the fifth day of incubation, the neutral protease activities of the beans and the mixed substrate were 14 and 15 unit/mL, respectively. In terms of the lipase activity, the mixed substrate resulted in a higher activity than the other substrates. Various volatile flavor compounds, including alcohols, ketones, esters, aldehydes, and pyrazines, were identified in R. oligosporus 'Koji' cultured using the mixed substrate.