AbstractStaphylococcus aureus is an opportunistic zoonotic pathogen which secretes 24 different types of enterotoxins (SEs) and enterotoxin‐like (SEls) proteins. Classical enterotoxins (SEA–SEE) are responsible for >95% of food poisoning outbreaks, of which SEA alone is responsible for >75% of them. This study was undertaken to develop a sandwich enzyme‐linked immunosorbent assay (ELISA) for sensitive, specific, and quantitative detection of staphylococcal enterotoxins‐A in food. Optimization of sandwich ELISA was attempted in two ways: rabbit polyclonal anti‐SEA as a capture antibody and mouse monoclonal anti‐SEA as a detector antibody, and vice versa. In the optimization of sandwich ELISA, mouse monoclonal anti‐SEA as a capture antibody and rabbit polyclonal anti‐SEA as a detector antibody yielded the highest sensitivity of 0.5–0.75 ng mL−1. The developed assay was found to be highly specific and exhibited equivalent sensitivity to a commercial kit. The developed sandwich ELISA may be utilized for the detection of staphylococcal enterotoxin‐A in food as a cheap alternative to available commercial kits. The developed sandwich ELISA may be useful for microbiological quality assurance of foods, especially in resource‐limited developing countries.