Monoclonal antibodies (MAbs) against Listeria monocytogenes were raised using the NCTC 11994 and EGD-e isolates of L. monocytogenes to immunize BALB/c mice. The antigenic protein of 65 kDa from L. monocytogenes EGD-e was found to react with MAb LMF3-238. This MAb characterized as IgG1 specifically reacted with 11 out of 12 different strains of L. monocytogenes, covering serotypes 1/2a, 1/2b, 1/2c, 4a and 4b that are virulent markers. MAb LMF3-238 detected L. monocytogenes EGD-e after inoculation into pork meat and storage at 4°C for 36 hours at a limit of detection of ~106 colony forming units/ml by dot-ELISA. From 311 Listeria spp. colonies, isolated from a total of 50 different fresh and processed foods and randomly selected from PALCAM agar after enrichment, L. monocytogenes was identified by MAb LMF3-238 using dot-ELISA with relative accuracy, relative sensitivity and relative specificity of 95.2%, 92.5% and 98.0%, respectively, compared to the multiplex-PCR method.