A mammalian cell-based optical biosensor was built to detect pathogenic <i>Listeria</i> and <i>Bacillus</i> species. This sensor measures the ability of the pathogens to infect and induce cytotoxicity on hybrid lymphocyte cell line (Ped-2E9) resulting in the release of alkaline phosphatase (ALP) that can be detected optically using a portable spectrophotometer. The Ped-2E9 cells were encapsulated in collagen gel matrices and grown in 48-well plates or in specially designed filtration tube units. Toxin preparations or bacterial cells were introduced and ALP release was assayed after 3-5 h. Pathogenic <i>L. monocytogenes </i> strains or the listeriolysin toxins preparation showed cytotoxicity ranging from 55% - 92%. Toxin preparations (~20 μg/ml) from <i>B. cereus </i>strains showed 24 - 98% cytotoxicity. In contrast, a non-pathogenic <i>L. innocua </i>(F4247) and a <i>B. substilis </i>induced only 2% and 8% cytotoxicity, respectively. This cell-based detection device demonstrates its ability to detect the presence of pathogenic <i>Listeria</i> and <i>Bacillus</i> species and can potentially be used onsite for food safety or in biosecurity application.