To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6-WS11, respectively. The strains WS3 (none) and WS4 (Delta nprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3-WS11, the pullulanase production levels of the resulting strains (WS3PUL-WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148.2 U/mL). Then, the scale-up pullulanase production levels of four recombinant strains WS5PUL, WS9PUL, WS10PUL, and WS11PUL were investigated in the 3-L fermenter cultivations. Strain WS9PUL produced the highest pullulanase activity (2449.6 U/mL) when fed an inorganic nitrogen source. However, the specific activity of the pullulanase obtained in a 3-L fermenter generally decreased as the number of protease deletions increased. Meanwhile, using pullulanase, alpha-cyclodextrin glucosyltransferase and beta-cyclodextrin glucosyltransferase as reporter proteins, the protein production differences among strains WS3, WS9, and the widely used WB600 were investigated. Finally, the carbon to organic nitrogen source ratio of the feeding solution used in the 3-L fermenter was optimized. Recombinant strain WS9PUL fed with carbon and organic nitrogen sources in a ratio of 4:1 achieved a pullulanase activity of 5951.8 U/mL, the highest activity reported to date.