Staphylococcus aureus rich in protein A when coated with monoclonal antibodies (MoAb) to Japanese encephalitis virus (JEV) gave a highly specific reaction with flavivirus antigens. The bacteria coated with JEV species-specific MoAb gave a strong co-agglutination with fifty-six JEV isolates from various parts of China, but no co-agglutination with Murray Valley encephalitis (MVE) and Kunjin (Kun) virus antigens. The flavivirus- and subgroup-specific MoAbs were reactive with MVE and Kun, as well as with the majority of the JEV strains. Blocking test with homologous MoAbs abolished co-agglutination further confirming its specificity. Numerous virus particles were observed on the surface of MoAb-coated staphylococci under the electron microscope after co-agglutination. The test appeared rapid, specific, simple to perform, and useful for rapid detection and identification of flaviviruses.