Objective To construct a recombinant strain expressing the antimicrobial peptide Plectasin, optimize the high-density fermentation conditions of the recombinant strain, and evaluate the antibacterial activity of the purified recombinant peptide produced under optimized conditions.Methods To improve soluble expression of the target protein, the plectasin gene was synthesized and optimized based on codon preference of the prokaryotic expression system with signal peptide deletion. The optimized gene was cloned into the pET32a(+) expression vector to construct the recombinant plasmid pET32a-plectasin, which was transformed into Escherichia coli BL21(DE3)/pLysS competent cells. Single-factor and orthogonal experiments were designed to optimize the parameters of 20 L high-density fermentation for the recombinant strain. The recombinant Plectasin produced under optimized conditions was purified and its antibacterial activity was analyzed.Results The E. coli BL21(DE3)/pLysS/pET32a-plectasin expression strain was successfully constructed. The optimized 20 L high-density fermentation conditions were induction temperature of 30 ℃, inoculum size of 2%, and fermentation duration of 15 h. After purification, the recombinant protein concentration reached (0.62±0.02) g/L. The minimum inhibitory concentration (MIC) of the expressed product against Staphylococcus aureus ATCC 25923 was 32 μg/mL.Conclusion Optimization of prokaryotic high-density fermentation conditions improved the soluble expression yield of recombinant Plectasin, and the fermentation product exhibited antibacterial activity against S. aureus.