Bacterial cell surface antigens interact with the host immune system resulting in the production of antibodies. Detection of antibodies against surface antigens has applications in diagnosis of many bacterial infections, assessment of immune status and epidemiological studies. We developed a microarray platform, for antibody detection, by printing Gram-negative and Gram-positive whole bacterial cells on nitrocellulose coated glass substrates. Antibody binding was detected using fluorophore labeled secondary antibodies. The sensitivity of antibody detection was found to be 0.1 microg/ml. Using bacterial cell microarrays it was also possible to successfully detect antibodies against Francisella tularensis in canine serum samples declared positive for tularemia based on microagglutination antibody titer. Use of bacterial cells as the antigen source in immunoassays has the advantages of simulating in vivo presentation of surface antigens and also eliminating the need for antigen purification. The microarray format gives the added advantage of simultaneous detection of antibodies against multiple bacteria employing only small amounts of samples and reagents.