The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis ofSARSis essential for the control of the disease in humans. RecombinantSARS-CoV S1 antigen was produced and purified for the development ofmonoclonaland bi-specificmonoclonal antibodies. The hybridomas secretinganti-S1antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coatingantibodyand biotinylated F26G18 as a detectionantibodywith a detection limit of 0.037μg/ml (p<0.02). The same detection limit was found with P136.8D12 as a coatingantibodyand biotinylated F26G18 as a detectionantibody. The sensitivity was improved (detection limit of 0.019μg/ml), however, when using bi-specificmonoclonal antibody(F157) as the detectionantibody. In conclusion, the method described in this study allows sensitive detection of a recombinantSARSspike protein by sandwich ELISA with bi-specificmonoclonal antibodyand could be used for the diagnosis of patients suspected withSARS.