Objective In order to construct a viral vector that can simultaneously express two non-fusion proteins in the whole host plants.Methods Agrobacterium infectious clone pYL156 containing tobacco rattle virus (TRV) genomic RNA2 was used to construct dual expression vector pTRV2e² by deleting 279 bp of 5" end of 2b gene, changing initiation codon of 2b gene ATG to AGG, and introducing the subgenomic promoter of pea early-browning virus (PEBV) coat protein (cp) gene. Different exogenous genes were cloned into the downstream of 2b and PEBV cp subgenomic promoters to measure the ability of virus TRVe² to express two foreign proteins, assess the stability of reconstructed TRVe² and analyze the function of proteins in the seedlings of Nicotiana benthamiana.Results TRVe² could simultaneously and rapidly produce two non-fused target proteins and express at least a 70 ku foreign protein in the whole host plants; TRVe² harboring 2.0 kb exogenous gene could stably exist in N. benthamina plants and could be served as technical means for analyzing the biological function of the proteins and the interaction between two proteins.Conclusion Recombinant virus TRVe² constructed in this study provide a toolbox for fast and efficient production of double foreign proteins and for analysis of the interaction between two proteins in the host plants.