Pullulanase is widely used in the starch processing industry as a debranching enzyme. However, extracellular production of pullulanase from recombinant Bacillus subtilis is limited and the loss of plasmids during fermentation of B. subtilis recombinants seriously affects the expression of the foreign protein, especially in large-scale production. In this study, a universal integrated plasmid was conducted harboring the pul cassette that included the pill gene encoding Bacillus naganoensis pullulanase (PUL), a constitutive promoter, P-H(pall), and an extracellular signaling peptide, LipA. This cassette was inserted into the genomes of B. subtilis WB800 B. subtilis WB600 by double homologous recombination. The pullulanase activity of up to 30.32 U/ml and 18.83 U/ml was achieved for B. subtilis WB800-P-Hpall and B. subtilis WB600-P-Hpall-pul, respectively, under primary conditions. To further enhance the yield of PUL, the effects of four important factors (inoculum size, incubation temperature, shaking speed, and initial pH) on the expression of PUL in shake flask fermentation were evaluated by "onefactor-at-a-time" technique for B. subtilis WB800-P(Hupall)pul. Consequently, the extracellular production of PUL was significantly enhanced, resulting in an activity of 60.85 U/ml.