Coxsackievirus B3 (CVB3) mainly infects the heart, and the timing and distribution of viral RNA polymerase (3D protein) expression in the myocardium are currently unclear. In this study, the CVB3 3D gene was obtained by polymerase chain reaction (PCR) and cloned into the prokaryotic expression vector pET28a(＋). The expression of 3D was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the 3D-His protein was purified and used to immunize New Zealand white rabbits for antiserum against the 3D protein. An anti-CVB3 3D antibody with high titer and specificity was obtained, and it can be used for future functional study of CVB3 3D protein.