Staphylococcal enterotoxins (SE) are the main causative agents of staphylococcal food poisoning. Unlike classical SEs (SEA to SEE), the relationship between newly identified SEs (SEG to SEX) and staphylococcal food poisoning has not been clearly elucidated. Recently, mild emetic potential of staphylococcal enterotoxin M (SEM) has been demonstrated, which indicated that SEM might be associated with food poisoning. However, there is currently no commercial enzyme-linked immunosorbent assay (ELISA) kit available for immunological detection of it. Therefore, we developed a highly sensitive, specific sandwich ELISA system and assayed SEM in food matrixes. This assay facilitates the study of SEM expression in vitro and in vivo. In addition, the investigations would be helpful in addressing the relative incidence of SEM+ strains in near future.