OBJECTIVE Constructing the recombinant Listeria ivanovii strain expressing green fluorescent protein to provide an important tool for study of Listeria ivanovii. METHODS The promoter of Listeria monocytogenes Listeriolysin O (phly) and the green fluorescent protein (GFP) gene were fused by SOEing PCR,and then ligated the fusion gene into plasmid pCW to result in recombinant plasmid pCW-phly-GFP. Recombinant plasmid was electroporated into Listeria ivanovii,and fluorescence microscope was used to analyze the expression of GFP. To observe the stability of recombinant plasmid and the stable expression of GFP in Listeria ivanovii,bacteria were cultured in the BHI broth with or without erythromycin for several generations. The stability of recombinant plasmid pCW-phly-GFP and fluorescent protein in each generation of bacteriawas studied by extracting plasmids and observing fluorescence. RESULTS The exactness of recombinant plasmid pCW-phly-GFP was confirmed with restrictive endonuclease assay and sequence analysis. Under the fluorescence microscope,the green fluorescence was obvious in Listeria ivanovii carried with pCW-phly-GFP. The recombinant plasmid pCW-phly-GFP was stable in Listeria ivanovii and the GFP kept expressing in a high level under the pressure of erythromycin. CONCLUSIONS The prokaryotic expression plasmid pCW-phly-GFP containing GFP gene was successfully constructed. Listeria ivanovii carried with the plasmid efficiently expressed GFP. This research provides an important tool for further study of Listeria ivanovii as a vaccine carrier.