Exotoxins secreted by Staphylococcus aureus have been identified as a possible trigger factor in atopic dermatitis (AD). We investigated the production and role of circulating antibodies, with specificity to staphylococcal enterotoxin B (SEB), in children with AD compared with those of healthy controls. The children with AD had significantly higher levels of serum SEB-specific immunoglobulin G (IgG; p = 0.0193), IgM (p = 0.011), and IgE (p = 0.0001) than the nonatopic children. The proportions of IgG, IgM, and IgE seropositivity in children with AD were 52.5% (21/40), 62.5% (25/40), and 67.5% (27/40), respectively. The levels of SEB-specific IgE and the severity of AD (p = 0.0004) were compared, but no correlation was seen for IgG or IgM. SEB may be involved in exacerbation of AD. SEB-specific IgE may be an important index of the clinical severity of AD. The SEB-specific IgG or IgM was produced during the exposure to the SEB antigen but may not be protective against SEB in AD.