In this study, we generated a gp90-specific mAb A9E8 using recombinant gp90 protein expressed in E. coli. The mAb A9E8 showed strong reactivity against purified whole virus in Western blot and could be used to detect REV-A antigen by an indirect immunofluorescence assay. The linear epitope recognized by the mAb A9E8 was defined as SVQYHPL by screening a random phage display peptide library. This peptide sequence was identical to 213 SVQYHPL 219 of the gp90 protein of REV-A. N-or Cterminal deletions of amino acids of this epitope demonstrated that 213 SVQYHPL 219 is the minimal requirement for recognition by A9E8. Fusion proteins GST-R1 with extended amino acid residues at the C terminus of the motif SVQYHPL showed increased binding activity compared with that of GST-H7wt in Western blot, indicating that alanine alone significantly increased binding activity of the core epitope to mAb A9E8. Thus, the peptide SVQYHPLA was determined to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8.