The objective of the study to determine the factors affecting the protease content generated during soybean fermentation by Bacillus subtilis. In this study, four factors were investigated during soybean fermentation: (1) pH, (2) fermentation temperature, (3) fermentation time and bacterial density, and (4) added glucose content on steamed soybean. The experimental results showed that at 33 °C temperature, 48 h fermentation time, bacterial density 104 CFU, pH 7, and supplementation of 1 g glucose into 50 g steamed soybean, Bacillus subtilis fermented soybean (another name is natto) had the highest protease activity. This suggests that the fermentation conditions in the study are consistent with the growth of Bacillus subtilis to produce proteases. Moreover, the optimization of fermentation conditions contributes to reducing the energy consumption to obtain the desired product, by increasing the yield in reaction. Therefore, this study is important at an industrial level, while contributing to the rational use of energy.