[Objective] We studied the function of glnA, proB and proA genes in the pathway of proline biosynthesis in Bacillus subtilis.[Methods] B. subtilis WB600 was used as the original strain. After a series of intracellular gene deletion and overexpression, we constructed recombinant strain WB601 with proB gene overexpression and WB602 with proA gene overexpression; WB603 with glnA gene deletion. Then, WB604 with proB gene overexpression was constructed. The anabolic key nodes were analyzed by phenotypes of extracellular and intracellular free proline accumulation.[Results] Under non-stress conditions, the extracellular proline content of the recombinant strains WB601 and WB602 were 2.21 times and 2.82 times of that of the original strain, the unit cell extracellular proline yield were 4.09 times and 9.80 times of that of the original strain, and the intracellular free proline content were 1.91 times and 3.34 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB603 increased to 1221.43 mg/L, 6.28 times of that of the original strain, and the unit cell extracellular and intracellular free proline yield were 9.13 times and 3.66 times of that of the original strain, respectively. The extracellular proline content of the recombinant strain WB604 up to 1391.65 mg/L, compared with strain WB603, the extracellular proline content and unit cell yield were increased by 13.94% and 14.10%, respectively, and the intracellular free proline content increased by 32.60%. Under 5% NaCl stress, the extracellular proline content of recombinant strains WB601 and WB602 were 1.94 times and 1.54 times of the original strain, and the unit cell yield were 2.15 times and 2.19 times of the original strain, respectively; the extracellular proline content and unit cell yield of recombinant strain WB603 were 4.16 times and 7.29 times of the original strain, respectively; under the same conditions, compared with the recombinant strain WB603, the extracellular proline content and unit cell yield of the recombinant strain WB604 were increased by 32.61% and 5.54%, respectively. In addition, the intracellular free proline content of the experimental groups were higher than that of non stress conditions, and reached a relative equilibrium state.[Conclusion] Overexpression of proB and proA gene can significantly enhance the ability of cells to synthesize proline and enhance the salt tolerance of cells; deletion of glnA could enhance proline de novo synthesis pathway and increase the accumulation of proline; moreover, the positive accumulation of the two effects could further improve the ability of proline synthesis.