Dengue virus (DENV) envelope protein is responsible for viral attachment to host cells and as such is a target of neutralizing antibody responses. However, the presence of envelope-specific antibodies against a given serotype may contribute to enhanced disease during secondary infection with another serotype. There is a need therefore for a standardized, high-throughput low-volume assay which permits the simultaneous screening of reactivity to multiple DENV serotypes. Here, we describe a method of identifying DENV serotype-specific response in exposed individuals using a multiplexed bead-based immunoassay. The ED3 domain of a specific DENV serotype is cloned into pQEAM containing 6xHIS, TEV protease, and AviTag biotinylation sites. Biotinylated ED3 proteins are expressed in E. coli CVB101 and purified by sequential column fractionation followed by coupling onto fluorescent avidin-coated microspheres. Methods for determining the optimum amount of biotinylated ED3 protein coupled onto the microsphere are described. The assay demonstrates both a high degree of sensitivity and specificity using well-characterized patient plasma samples. The nature of the assay permits further development to include a variety of DENV serotypes and regionally important sub-serotypes.