Abstract: In order to address the problems of solid-state fermentation of nattokinase, such as uncontrollable conditions, low enzyme production, and poor flavor,the optimization of culture medium, compounding of grains and fermentation at different temperatures were investigated in this study. Firstly, the solid-state fermentation medium and fermentation conditions were optimized by single-factor and response surface. Secondly, the enzyme yield and flavor were further improved by soybean crushing, grain compounding, and temperature-change fermentation. Then, the samples were analyzed for volatile flavor substances and volatile saline nitrogen. Lastly, the isoflavones content was determined by high-performance liquid chromatography (HPLC) and analyzed for antioxidant activity. The results showed that when using soybeans as the raw material, with an inoculum amount of 5.8%, sucrose at 2.18%, tryptone at 1.51%, KCl at 0.1%, and fermentation at 30 ℃ for 24 h, the nattokinase activity was 9924.81 IU/g, and the enzyme production increased by 81.87%. When soybeans were split into halves and compounded at a soybean-to-soybean meal mass ratio of 4:1, fermentation at 30 ℃ for 24 h followed by transfer to 40 ℃ for 8 h resulted in an enzyme activity of 18453.77 IU/g, representing a 238.17% increase over pure soybean fermentation. Compounding with grains weakened the off-flavor and reduced volatile basic nitrogen by 17.22%. Meanwhile, the fermented samples with grain compounding showed increased active components, with soybean isoflavones and total antioxidant capacity rising by 39.95% and 20.60%, respectively. It is demonstrated that effective improvement of nattokinase yield and active components, along with reduction of fermentation-derived peculiar smells, can be achieved through adjusted raw material pretreatment and optimized fermentation conditions, providing a theoretical basis and technical support for efficient nattokinase production.