[Objective] The study was to clone, express the gene of chicken antimicrobial peptide fowlicidin-2 and characterize its bioactivity. [Methods] The gene encoding fowlicidin-2 with the condon preference of E. coli was designed based on the amino acids of fowlicidin-2 and synthesized in vitro. The gene was ligated into the plasmid pET32a, and transformed into E. coli BL21(DE3). The transformant E. coli BL21(DE3) was induced by IPTG. Cyanogen bromide (CNBr) was used to cleave the inclusion body of fusion protein and molecular sieve chromatography was used to purify the released fowlicidin-2. Bioactivity of the recombinant fowlicidin-2 was tested. [Results] The fowlicidin-2 fusion protein was expressed as inclusion body. Fowlicidin-2 was effectively released after digestion of the fusion protein with CNBr. The recombinant fowlicidin-2 exhibited high antibacterial activity against the Gram-positive and Gram-negative bacteria. [Conclusion] Fowlicidin-2 was successfully expressed in E. coli. The study provides theoretical foundation and technical means for scale-up preparation of antimicrobial peptides by engineering method.