A molecular beacon-based real-time NASBA (QNASBA) assay for detection and identification of Listeria monocytogenes has been developed. A correlation between targeting highly accessible mRNA sequences and QNASBA efficiency and sensitivity was demonstrated. The assay targets a sequence from the mRNA transcript of the hly gene which is specific for this bacterium; and includes an internal amplification control to disclose failure of the reaction. It was fully selective and consistently detected down to 100 target molecules and 40 L. monocytogenes exponentially growing cells per reaction. In addition, it was capable of accurate quantification of target RNA molecules independently of the presence of DNA in the sample. In combination with a short RNase treatment prior to nucleic acids extraction our QNASBA specifically detected viable L. monocytogenes cells. It was successfully applied to rapid detection of this pathogen in meat and salmon products, and is therefore a useful tool for the study of L. monocytogenes in food samples. We finally discuss considerations of target secondary structure with regard to development of NASBA assays.