Johne's disease (JD) in ruminants is caused by the bacterium Mycobacterium avium subsp. paratuberculosis (MAP). JD has caused significant economic loss in affected farms. The worldwide distribution of MAP and its potential link to Crohn's disease in humans raises a public health concern. To facilitate more widespread acceptance of JD control programs, inexpensive diagnostic method that requires less time, inexpensive equipment and that are user friendly must continue to be developed. Diagnosis of JD is done by either detecting MAP in feces or infected tissues or detecting an immune response. Common assays used to detect MAP antibodies include: compliment fixation (CF), agar gel immunodiffusion (AGID), and enzyme linked immunoabsorbent (ELISA) assays. In this study, a polyaniline-based conductometric biosensor was developed for the rapid detection of antibodies to MAP in cattle serum. The conductometric biosensor consists of three components; the biological sensing element, the transducer and the detector element. MAP antigen served as the biological sensing element, polyaniline (Pani) was the transducer and an ohmmeter was used as the detector element. The biological element and the transducer are immobilized in a membrane with four regions; sample application, conjugate, capture and absorption regions. The detection technique is based on the antigen-antibody coupling reaction with Pani as the conductive transducer. Pani is conjugated to the heavy chains of the monoclonal anti-bovine IgG (AB/IgG) forming Pani-AB/IgG, which is positioned in the conjugate region. MAP purified proteins, serving as capture antigens; are immobilized on the nitrocellulose capture region. Subsequently, silver electrodes are fabricated on both sides of the capture region. 0.1 ml of the sample is added to the application region and allowed to flow through the other regions through capillary movement. Theoretically, Pani-AB/IgG in the conjugate region binds to the bovine IgG present in the sample. The conjugated bovine IgG-Pani-AB/IgG flow through the capture zone where MAP specific antibodies are captured by immobilized MAP purified proteins. As MAP specific antibodies are captured, the attached Pani structures form a bridge across the electrodes flanking the capture region. This bridge closes an electrical circuit that can be measured as a decrease in electrical resistance as more antibodies are captured. In this proof of concept study, JD positive cattle sera, as determined by standardized ELISA technique, were compared to serum samples that were negative for JD. The average resistance for JD negative samples was 92.875 kΩ (Kilo ohms) (range 101.3-72.1 kΩ) while the average resistance for JD positive samples was 53.338 kΩ (range 66.4-37.0 kΩ). These initial studies demonstrate that a conductometric biosensor can be fabricated to detect antibodies to MAP. Further testing to optimize the biosensor performance and test with large numbers of samples is underway.