A bi-cistronic baculovirus transfer vector was constructed based on the 5'UTR internal ribosome entry site (IRES) of the Rhopalosiphum padi virus (RhPV). Recombinant baculoviruses containing the red fluorescent protein gene and green fluorescent protein gene flanking the RhPV 5'UTR IRES can simultaneously produce dual fluorescence in recombinant virus-infected Spodoptera frugiperda 21 cells (Sf21) under the control of a polyhedrin promoter. Quantization by fluorescence spectrophotometry of the fluorescent proteins produced in Sf21 cells indicated that the translational efficacy of the RhPV 5'UTR IRES was about 3-fold weaker than cap-dependent translation. We also demonstrated that recombinant baculoviruses containing the human interferon-gamma gene (IFN-gamma) and green fluorescent protein gene flanking the RhPV 5'UTR IRES can produce IFN-gamma proteins as well as green fluorescent proteins. These results suggest that the RhPV IRES can be used in the development of bi-cistronic baculovirus expression vectors for production of heterologous multiprotein complexes or can be combined with selection markers to facilitate applications of baculovirus expression systems.