The fusion expression strategy of human beta defensin 3(hβD-3) gene in Escherichia coli cells in the fusion expression by pET-32a(+) vector was studied. Fusion expression of F-βD-3 protein was performed in lysogenic bacterium BL21(DE3) by pET-32a(+) vector. The results showed that the production of F-βD-3 protein was 127.23 μg/mL. After nickel column chromatography and enterokinase cleavage, the fusion protein production finally obtained was 18.17 μg/mL. Recombinant hβD-3 protein products were identified to possess inhibitory activity by bacteriostatic test on E. coli K₁₂D₃₁.