We report a new, suspended-microsphere diagnostic test to detect antibodies to West Nile (WN) virus in human serum and cerebrospinal fluid (CSF). The microsphere immunofluorescence assay can be performed in less than 3 h on specimens of ≤30 μl. A recombinant WN virus envelope (E) protein antigen is covalently coupled to fluorescent polystyrene microspheres. After incubation with diluted serum or CSF, antibodies bound to the E protein antigen are detected with fluorescently labeled anti-human immunoglobulin antibody and flow analysis in a dual-laser Luminex 100 instrument. Retrospective testing of 833 sera from New York patients with suspected viral encephalitis demonstrated concordance with results obtained with the traditional enzyme-linked immunosorbent assay for immunoglobulin G (IgG) antibodies to WN virus (kappa = 0.85). One hundred eighty-eight (22.4%) of the samples, which were collected from June to November 2002, tested positive for antibodies to WN virus in the microsphere assay. Specimens depleted of IgG with anti-IgG antibody were reassayed to measure anti-E protein IgM antibodies and to provide an indication of current or recent WN virus infection. The assay also detects antibodies to E proteins from related flaviviruses, including St. Louis encephalitis, Japanese encephalitis, and dengue viruses. The new microsphere immunoassay provides a sensitive and rapid alternative to traditional enzyme-linked immunosorbent assays that detect antibodies to flavivirus E proteins. This assay can aid physicians and public health workers in the management of outbreaks of WN virus and related flaviviruses.