In order to optimize the processing technology of natto, improve the flavor of the product, and to explore the effect of double-bacteria mixed fermentation on the quality of natto, two better strains were screened through the ratio of the diameter of the hydrolytic colonies (C/H), growth curve of the strains, nattokinase activity and volatile base nitrogen content. The single-factor and orthogonal experiments were used to optimize the process conditions of the two-bacteria fermentation of natto, with nattokinase activity and volatile base nitrogen content as the indicator. The optimal fermentation process of natto with two strains is the ratio of strains (m_N5∶m_N3) 4∶1, inoculation volume 9%, fermented for 25 h at 35 ℃. Natto prepared under this fermentation condition has a nattokinase activity of 481.84 U/g, which increased by 36.8% compared with single-bacteria fermentation; the volatile base nitrogen content was 205.21 mg/100 g, which was reduced by 42.3%, compared with the highest value of single fermentation. The results showed that using two strains of Bacillus natto to optimize the fermentation process could provide a way to enhance enzyme activity and improve the flavor.