By using bioinformatics software,the domain of antigenic epitopes on Tobacco ringspot virus (TRSV) coat protein was analyzed and predicted,and the epitopes were found to be located near 319-328,384-388,420-430,480-487，495-511 amino acid residues at C-terminal. After the targeted fragment was amplified by RT-PCR based on the specific primers, the expression vector was constructed and the monoclonal antibody was prepared with the fusion protein and hybridoma. The antiserum prepared with the fusion protein reacted specifically with the expressed product of TRSV coat protein in E.coli. The antiserum had high specificity and may be applied to quarantine and inspection.