Development and validation of a mammalian cell-based biosensor for application in food defense and food safety was investigated. Three prototypes of the biosensor capable of handling different sample types were developed and tested with food and beverages. The sensing element is a B lymphocyte Ped-2E9 cell-line, encapsulated in collagen matrix in 3D scaffold. The uniqueness of this biosensor is that it detects analyte interaction with mammalian cells and is able to distinguish pathogenic from non-pathogenic and active from inactive toxins, rendering accurate estimation of the risk associated with the agents. This sensor gave positive signal for a broad range of bacterial pathogens; Listeria monocytogenes, enterotoxigenic Bacillus, Vibrio, Micrococcus and Serratia, and toxins; α-hemolysin from Staphylococcus aureus, phospholipase C from Clostridium perfringens, cytolysin from sea anemone Stoichactis helianthus, listeriolysin O from L. monocytogenes, and enterotoxin from Bacillus. Detection limit for toxins was 10-40 ng in 2 h while for a model bacterial pathogen, L. monocytogenes, 10(3)-10(4) CFU/ml in 4-6 h, even in the presence of a mixture of higher concentrations of non-pathogenic species of the same genera or common background microflora. With inoculated food and beverage, the sensor detected L. monocytogenes and Bacillus cereus at a low initial concentration of 10(2)-10(4) CFU/g from ready-to-eat meat and rice, and only active toxins at nanogram quantities from rice, milk and water samples. Though all the three prototypes performed well with beverages, Devices II & III are most suitable for testing particulate foods. These data present promising evidence for possible application of this biosensor for rapid detection of multiple pathogens or toxins for food defense and food safety application.