Acrylamide, a toxic and carcinogenic compound, has been found to be present in a range of processed starchy foods. To prepare an effective immunogen compound for acrylamide, N-acryloxysuccinimide (NAS) was conjugated to bovine serum albumin (BSA) at a high molar ratio of 21.2 : 1. Antisera were obtained by immunization of rabbits with additional booster injections of the NAS–BSA conjugate after the regular process. The IgGs purified by an ammonium sulfate precipitation method were further fractionated with a BSA-immobilized immunoaffinity column. The affinity constant between the collected antibody and coated antigen (NAS–ovalbumin) is found to be 6.7 × 10⁷ L mol⁻¹. Asparagine, the key precursor of acrylamide formation in foods, showed negligible cross-reactivity to the antibody. A biotin–avidin enzyme-linked immunosorbent assay (BA-ELISA) was developed and the optimum assay medium was found to be 0.1 mol L⁻¹NaHCO₃ (pH 8.3, containing 0.5 mol L⁻¹NaCl). The BA-ELISA afforded a practical sensitivity with a working range of 10–100 000 ng mL⁻¹ and a detection limit of 6 ng mL⁻¹. The assay was applied to detect acrylamide in potato fries and biscuits and the quantitative results were in good agreement with those obtained by the high-performance liquid chromatography method. This immunoassay will be very useful for monitoring acrylamide in food samples.