Listeria monocytogenes is the third most deadly foodborne pathogen in the United States. The bacterium is found in soil and water, contaminating raw food products and the processing environment, where it can persist for an extended period. Currently, testing of food contact and non-food contact surfaces is performed using an array of sampling devices and endpoint technologies, offering various levels of sensitivity, cost, user skill, and time to detection. Paper-based microfluidic devices (µPADs) are a rapid detection platform amenable to low-cost, user-friendly, and portable diagnostics. In this study, we developed and evaluated a µPAD platform specific for the colorimetric detection of the Listeria genus following recovery from food contact and non-food contact surfaces. For detection, four colorimetric substrates specific for the detection of β-glucosidase, two broths selective for the detection of Listeria spp., and a nonselective broth were evaluated to facilitate detection of Listeria spp. The limit of detection and time to detection were determined by using pure bacterial cultures. After 8 h enrichment, L. monocytogenes (102 Colony Forming Units (CFU)/coupon) was detected on every surface. After 18 h enrichment, L. monocytogenes (102 CFU/coupon) was detected on all surfaces with all swabbing devices. This study demonstrated the ability of the µPAD-based method to detect potentially stressed cells at low levels of environmental contamination.