Human neutrophil peptides (HNPs) possess high antimicrobial activities against a broad spectrum of microorganisms. Rhodobacter sphaeroides is the best-characterized photosynthetic bacterium and exhibits potential as a novel expression system. Up to date, no literature has been reported regarding expression of HNP3 in Rb. sphaeroides. In the present study, the HNP3 gene fragment was amplified by SOE PCR and ligated into photosynthetic bacteria light-harvesting complex 2 (LH2) expression vector leading to HNP3 fusion protein expression vector. The HNP3 fusion protein was successfully expressed as rapidly evaluated by the LH2 characteristic peaks at ~800 nm and ~850 nm before purification and SDS/PAGE. Subsequently, the HNP3 fusion protein was purified by one-step affinity chromatography, and could be rapidly detected by the color and the spectral absorption at ~800 nm and ~850 nm before SDS/PAGE. Antimicrobial activity assay suggested that the HNP3 fusion protein exhibited high antimicrobial activity towards E. coli. The present study may supply an insight into employing the novel Rb. sphaeroides expression system, exhibiting dramatic advantages over currently used commercial expression system, to heterologously express human neutrophil peptides.