Objective To construct a green fluorescence protein (GFP) adenovirus vector specifically labeled with peroxisomes (Ad-GFP-Peroxi). Methods The GFP sequence modified with peroxisome signal peptide sequence was designed. The sequence fragment was amplified by polymerase chain reaction and linked with adenovirus shuttle plasmid vector (pAdTrack); then the connecting product was transformed into E. coli DH5α. The recombinant plasmids were obtained from DH5α cells and sequenced for identification. The recombinant plasmid was linearized by PmeⅠ, and homologous recombination was carried out in BJ5183 competent cells containing pAdEasy-1. The recombinant plasmid was linearized by PacⅠ, and then used to infect 293A cells for virus packaging to obtain Ad-GFP-Peroxi particles. Rat cardiomocytes H9C2 were infected with Ad-GFP-Peroxi adenovirus or Ad-GFP adenovirus, and the green fluorescence intensity was observed under fluorescence microscope after 48 h. H9C2 cells infected with Ad-GFP-Peroxi adenovirus were divided into 2 groups (normal culture group and 1% oxygen concentration group), observed under confocal microscope after 24 h, and the fluorescence signal aggregation was analyzed. Results The recombinant Ad-GFP-Peroxi was successfully constructed. Compared with Ad-GFP, Ad-GFP-Peroxi could specifically display the distribution of peroxisomes in H9C2 cells. Peroxisomes accumulated in H9C2 cells after hypoxia stimulation. Conclusion Ad-GFP-Peroxi is successfully constructed by homologous recombination, and it can highly infect rat cardiomyocytes H9C2, clearly showing the distribution of peroxisomes in cardiomyocytes under confocal microscope.