In economic terms, the microbiological safety of food is a serious concern of consumers and the food industry owing to the significant public health and global economic impact of foodborne diseases. Thus, specific and sensitive detection of pathogenic bacteria is required to ensure food safety. In this study, we constructed a novel paperbased nucleic acid diagnostics for viable pathogenic bacteria detection with  naked eyes. An effective isothermal amplification method was described to amplify the hlyA mRNA gene of Listeria monocytogenes. The amplification products were applied to the paperbased platform to perform a visual test without prehybridization. When the DNA products migrated along the platform by capillary action, the goldnanoparticles accumulated at the designated area. Under optimized experimental conditions, as little as 100 pg/μL total RNA could be detected. The whole assay process, including ligation, amplification, enzyme digestion, and detection, can be completed within several hours. This method is suitable for pointofcare applications to detect foodborne pathogens. Furthermore, this study highlights the potential of the paperbased nucleic acid diagnostics platform for cost and laboreffective applications in the detection of pathogenic bacteria.