To produce tachyplesin, an antimicrobial peptide, by a stable and efficient gene engineering approach, cDNAs containing single tachyplesin gene sequence (tac)(1) and tandem repeat of tachyplesin gene sequence (tac2) were respectively developed by annealing two synthesized complementary single-stranded DNAs and constructed into pSBPTQ shuttle vector under the control of the SacB.p.s promoter. The vectors containing the target gene sequence were then transformed into Bacillus subtilis WB800, respectively. Both expression of tac and tac2 were induced by 2% sucrose. The fermentation supernatant was purified by regenerated cellulose membrane tubing (MWCO 2000) and the secreted TAC(2) and TAC2 were about 5 and 10 mg/l of supernatant, respectively. The antimicrobial activities of TAC and TAC2 were measured by the size of bacteriostatic circle of the fermentation supernatants against Escherichia coli K88. Ultrastructural alteration of E. coli K88 and Salmonella typhimurium was observed under scanning electron microscope and transmission electron microscopy. The results showed that in comparison with TAC, TAC2 was expressed at a higher level and also indicating strong antimicrobial activity both in vitro and in vivo.