In this study, a sandwich ELISA was developed to address both rapidity and specificity in order to detect the p60 protein secreted by Listeria monocytogenes, a harmful foodborne bacterial pathogen. By sequentially combining the use of a monoclonal antibody against a L. monocytogenes specific 11-amino acids peptide sequence on the protein p60 and polyclonal antibodies against the whole p60 protein, 103 CFU/ml of L. monocytogenes were specifically detected without cross reaction with Listeria innocua, Escherichia coli, Salmonella Typhimurium and S. aureus. The detection was preceded by an incubation period of 18 h with minimal experimental manipulations. Among the sample preparation procedures tested, samples directly from L. monocytogenes bacterial culture allowed better detection as compared to cell-free supernatant samples. This sandwich ELISA is an experimental design that could be easily adapted for use in food processing industries for routine monitoring of washing and sanitizing procedures.