A single tube detection kit was designed as a rapid, easy-to use and reliable test to detect Listeria spp.. Various food samples (vegetables and raw catfish fillets) were used in order to validate the performance of the detection kit. L. grayi was detected in one ready-to-eat (RTE) vegetables with the detection kit while no Listeria spp. was detected using the modified FDA-BAM method. In addition, both the detection kit and modified FDA-BAM method indicated that twelve catfish fillets were Listeria positive. The detection kit had 100% sensitivity and specificity in less detection time (24 h) than the modified FDA-BAM method (60% specificity, >72 h). There was no difference (P<0.05) between the kit and the modified FDA-BAM method on MPN for Listeria spp.