A capture monoclonal antibody-based assay has been established for detecting the p24 core protein and the gp51 envelope glycoprotein of bovine leukemia virus (BLV). This assay is rapid, highly sensitive and specific. Viral antigens in test samples were identified using mouse monoclonal antibody-coated or microtiter plates by adding labeled monoclonal antibodies with different epitope specificities. The choice of an appropriate epitope specificity for the specificity of monoclonal antibodies was important for optimal performance of the assay. Results of this assay were in agreement with the syncytia induction assay routinely used for detecting BLV production by cells in vitro. The sensitivity of monoclonal antibody assay was 0.5 ng/ml for p24 and 1.25 ng/ml for gp51, respectively. The specificity was demonstrated by immunoblotting. The assay can be performed in a few hours, is simple, and is comparable with more time-consuming assays with regard to sensitivity and specificity.