Immunological detection of viruses and their components usingmonoclonal 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) ofSARS-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/cmicewere 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, recombinantSARS-CoV N protein was expressed and purified and nine specific MAbs againstSARS-CoV N protein were obtained successfully. This panel ofanti-N MAbs may be used as a tool for rapid and specific diagnosis ofSARS-CoV.