Enteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example， EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis， which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development， methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybridoma clones were selected based on their reactivity in different immunoassays. The most promising clone， 3A6， was characterized and it performed well in multiple techniques including ELISA， immunoelectron microscopy， immunocyto- and histochemistry and in Western blotting， detecting EVs in infected cells and tissues. It recognized several EV-Bs and also the EV-C representative Poliovirus 3， making it a broad-spectrum EV specific antibody. The 3A6 rat monoclonal antibody can help to overcome some of the challenges faced with commonly used EV antibodies: it enables simultaneous use of mouse-derived antibodies in double staining and it is useful in murine models.