To determine the amounts of staphylococcal enterotoxin A (SEA), a novel and sensitive enzyme-linked immunosorbent assay (ELISA) was developed. Protein A, which is produced by Staphylococcus aureus, interferes with the reaction between SEA and anti-SEA immunoglobulin G (IgG), resulting in a false-positive reaction. Chicken IgY was introduced as a capture antibody in the sandwich ELISA system, as IgY binds less efficiently to protein A. When the anti-SEA IgG antibody was used as the capture and detection antibodies (IgG-IgG ELISA), the background levels of protein A increased, thus resulting in a false-positive reaction. A 0.01 ng mL−1 concentration of protein A significantly increased the absorbance value of the blank wells. When the anti-SEA IgY antibody was used as the capture antibody, 1000 ng mL−1 of protein A did not affect the absorbance value. The ELISA system using anti-SEA IgY as a capture antibody and anti-SEA IgG as a detection antibody (IgY-IgG ELISA) showed a detection limit of <0.25 ng mL−1 and a creditability of R2 = 0.98. These findings demonstrate the advantage of chicken IgY for the detection of SEA by means of double-antibody sandwich ELISA.