To achieve efficient expression and secretion of a biologically-active pullulanase, the effect of promoter and signal peptide on the production of pullulanase was studied. Three types of promoters (P-P43, P (apr) and P (amy) ) and four types of signal peptides (SP (sacB) , SP (amy) , SP (aprl) and SP (aprs) ) were combined to construct twelve expression cassettes for pullulanase in Bacillus subtilis. The pullulanase activity assay was employed to quantify the level of differential expression, and a real-time PCR assay was applied to comparatively track the transcriptional level. Under the same experimental conditions, the potency ratios among the three promoters were P (apr) > P (amy) > P-P43. The secretion efficiency ratios mediated by the signal peptides were SP (sacB) > SP (amy) > SP (aprs) > SP (aprl) . The highest yield of pullulanase could be achieved under the promotion mediated by P (apr) and secretion by SP (sacB) .