Nattokinase, traditionally produced by the fermentation of Bacillus subtilis natto, has high-efficiency and safe thrombolytic effect. However, low activity limits its industrial application as thrombolytic agent. In this study, the nattokinase encoding-gene from B. subtilis natto was heterologously expressed in B. subtilis WB800 to realize the high-efficiency of nattokinase. At first, the gene was amplified and connected to the Escherichia coli-B. subtilis shuttle plasmid pP43NMK to construct the recombinant plasmid pP43NMK-NKF. The recombinant WB800-NKF showed nattokinase enzyme activity of 22.3 FU/mL, with a molecular weight of enzyme protein of 27.7 kDa by SDS-PAGE. The enzyme activity increased to 75.3 FU/mL after the signal peptide starting codon is optimized as ATG from GTG. A total of 19 signal peptides were screened to construct a series of recombinants (PSP1–PSP19) and compared with the original signal peptide of nattokinase. Among them, the recombinant PSP7 (SPYqxM) showed the highest nattokinase enzyme activity, 235.2 FU/mL. Furthermore, the construction of the promoter tandem expression vector proved that the dual-promoter recombinant PSP7C43 (P43-P43) has the highest enzyme activity of 247.1 FU/mL, 1.16 times that of the single promoter recombinant PSP7 (P43). After the optimization of fermentation conditions, the nattokinase enzyme activity of recombinant PSP7C43 finally increased to 325.4 FU/mL. This enzyme activity reached the highest level under the same detection method. The combinatorial strategy adopted in this study realizes the substantial improvement of nattokinase expression level, which lays a foundation for the industrial development of nattokinase as a thrombolytic reagent.