Immunological detection of viruses and their components using monoclonal antibodies (MAbs) is a powerful diagnostic method. Here we report a detailed method for the establishment of MAbs against severe acute respiratory syndrome coronavirus (SARS-CoV). To express and purify the nucleocapsid protein (N protein) of SARS-CoV and generate MAbs against the N protein, gene encoding N protein was separated into two parts according to the prediction of epitopes and cloned into pET32a(+), respectively. Expression of the target proteins were induced by M isopropyl-β-thio-D-galactopyranoside (IPTG) and purified by a single-step affinity chromatography on a Ni-NTA column. BALB/c mice were immunized with the purified recombinant proteins to prepare MAbs by hybridoma technique. The reactivity and specificity of the MAbs were analyzed by ELISA and Western blot analysis. Seven MAbs against N1 and two MAbs against N2 were obtained. In the present study, recombinant SARS-CoV N protein was expressed and purified and nine specific MAbs against SARS-CoV N protein were obtained successfully. This panel of anti-N MAbs may be used as a tool for rapid and specific diagnosis of SARS-CoV.