A capture enzyme-linked immunosorbent assay (ELISA) for the identification of Listeria monocytogenes in food was standardised and validated. The assay was refined by analysing samples of meat, seafood, dairy products, pasta and flour. The method was found to be 100% specific for Listeria spp. tested, with a limit of sensitivity of 6.6 × 10(3) colony-forming units (cfu)/ml. Comparison of L. monocytogenes capture ELISA against the official International Organization for Standardization (ISO) method 11290-1:1996 for the isolation and identification of L. monocytogenes in food matrices produced a significant concordance index. The assay was validated on food matrices including meat, seafood and dairy products in line with ISO 16140:2003 concerning qualitative analytical methods. The assay was found to be accurate, specific, sensitive, selective, reproducible and fast, resulting in lower costs and faster turnaround in microbiological screening of foods.