A broad-spectrum monoclonal antibody (C4 MAb) against the capsid proteins (CPs) of plant potyviruses has been generated in previous studies. To clarify which epitope is recognized by this MAb, epitope mapping was performed via phage display library screening and amino acid substitution analysis. Subsequently, a 12-residue epitope in the core region of potyvirus CPs was identified and termed the C4 epitope (WxMMDGxxQxxY/F). This epitope contains tryptophan and tyrosine residues that are crucial for reacting with C4 MAb. The CP of Odontoglossum ringspot tobamovirus (ORSV) separately fused with the C4 epitope of Konjak mosaic potyvirus (KoMV), Zantedeschia mild mosaic potyvirus (ZaMMV), or Dasheen mosaic potyvirus (DsMV) was expressed in a bacterial system and purified. The results of indirect ELISA and Western blotting demonstrated that the C4 epitope of KoMV (Ko) fused to ORSV CP showed the strongest binding affinity to C4 MAb among the three viral epitope tags examined. The binding affinity between Ko tag (WTMMDGEEQIEY) and C4 MAb was determined. To examine the applicability of the Ko tag in planta, GFP and ORSV CP were transiently expressed in Nicotiana benthamiana, and both Ko-tagged proteins were specifically detected using C4 MAb. The Ko tag did not affect the silencing suppressor function of Tomato bushy stunt tombusvirus P19 in N. benthamiana. Furthermore, Ko-tagged EGFP could be successfully expressed, specifically detected and subsequently immunoprecipitated using C4 MAb in a mammalian cell system. Thus, the present study identified a common C4 epitope of potyviruses recognized by the broad-spectrum C4 and PTY 1 MAbs, and the results indicated that the newly designed Ko tag is suitable for application in bacterial, plant, and mammalian cell systems.