A novel DNA extraction and real time fluorescent polymerase chain reaction assay has been optimized for the detection of Mycobacterium avium paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting a specific sequence within the IS900 gene of MAP. In this study, DNA was successfully extracted by utilizing the Whatman FTA® card technology, which allows for simple processing and storage of samples at room temperature. The Whatman FTA® cards are washed and subjected to a Chelex -100 incubation to remove any remaining PCR inhibitors and to elute the DNA from the FTA® card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Highly specific detection of MAP DNA from bovine fecal samples spiked with known concentrations of MAP was obtained. The detection limit of the assay was consistently found to be 100 CFU/g but some samples containing 10 CFU/g could also be detected.