Human interferon-beta (hIFN-beta) was used as a heterologous model protein to investigate the effects of the Bacillus subtilis AmyE propeptide and co-expression of PrsA in enhancing the secretion of heterologous proteins in B. subtilis. Secretion and activity of hIFN-beta with AmyE propeptide increased by more than four-fold compared to that without AmyE propeptide. Moreover, under conditions of co-expressed PrsA, the secretion production and activity of hIFN-beta with AmyE propeptide increased by more than 1.5-fold. AmyE propeptide and co-expression of PrsA thus have an additive effect on enhancing the production of the hIFN-beta in B. subtilis.