Coxsackievirus B3 (CVB3) is the nonenveloped virus containing a single-stranded positive-sense RNA as a genome. CVB3 infection can induce acute myocarditis and dilated cardiomypathy. CVB3 of icosahedral symmetry has four capsid proteins called VP1, VP2, VP3, and VP4. Although VP1 is a major antigenic determinant, VP2 is also an important protein for viral physiology, such as maturation cleavage and attenuation. However, VP2 study has been hampered, partly because VP2 antibody is not available. In this study, we developed peptide-based polyclonal VP2 antibody and analyzed its potency by Western blotting analysis and immunofluorescent assay. Purified B3-1 antibody (VP2 peptide antibody developed in here) showed the sensitivity and specificity, similar to VP1 monoclonal antibody which is commercially available. Moreover, this peptide antibody may be useful for double-staining with other antibodies derived from mouse. Therefore, the VP2 antibody may allow us to study CVB assembly and understand VP2 function in depth.