Nucleocapsid (N) protein of virus plays special roles in stabilization of viral genome, regulation of viral replication and adapting cellular circumstance. Murine hepatitis virus (MHV), the prototype of Betacoronavirus, is a classic model for the exploration of coronavirus N protein functions. Viral N proteins from detergent-treated virions and recombinant N proteins expressed in prokaryotic cells were prepared and used to immunize BALB/c mice for the preparation of polyclonal and monoclonal antibodies respectively. Antibodies with high sensitivity and specificity against the antigens were selected and subjected an assay for antigenic determinants. The results indicated that both polyclonal antiserum and mAb 2E6 recognized a region covering 58 residues motif. An enzyme-linked immunosorbent assay (ELISA) with mAb 2E6 was established for N proteins in medium supernatants and cellular lysates. The signal could be detected at 4 h after infection and the readings were consistent to viral titers.