An absorption-inhibition ELISA (AI-ELISA) was developed to detect, within an hour, the staphylococcal enterotoxin B (SEB) in solutions and food homogenates. The SEB-containing sample is first incubated with a standardized concentration of antibody. The quantity of nonabsorbed anti-SEB Ab is evaluated by incubating a SEB-coated dipstick in the reaction mixture. The protein A-peroxidase conjugate is then used to detect the anti-SEB Ab bound to SEB-coated dipsticks. The enzymatic activity developed in a solution containing the substrate and a chromogen is inversely related to the SEB concentration. Concentrations as low as 0.01 to 1 μg SEB/ml can be consistently detected within 20 min. In unconcentrated food homogenates, 100 ng of SEB per ml could be detected by this AI-ELISA. The minimal level of sensitivity could be lowered to at least 1 ng SEB/g of food. The rapidity of the AI-ELISA, its simplicity, and repeatability make this immunoassay a promising tool for routine detections of staphylococcal enterotoxins in foods.