Escherichia coli O157:H7, an emerging cause of food-borne disease with the occurrence of an estimated 20,000 illnesses and 250 deaths each year in the United States, has now been reported from several countries worldwide. Infections with this bacteria, which follows the ingestion of contaminated food by humans, causes bloody diarrhea, hemolytic uremic syndrome (HUS), and renal disease, that can have serious health implications. The source of food contamination is usually associated with animals, mainly cattle. Many cattle become infected early in life when they are exposed to an environment that is contaminated by other animals shedding the organisms in their feces. Detection of E. coli O157:H7 in feces or contaminated food samples requires tests with high sensitivity, which is increased by the use of monoclonal antibodies. However, the production of concentrated monoclonal antibodies in ascites raises animal welfare concerns, and can be expensive. In this study, single chain of variable fragment (scFv) molecules were developed from hybridoma clones that produce immunoglobulins specific for the LPS and flagella antigen of E. coli O157:H7 using phage display technology. The reactivity of the soluble scFv for their respective antigens was preserved in ELISA and by partial inhibition of bacterial agglutination with polyclonal antiserum. Furthermore, the scFv were able to capture E. coli O157:H7 bacteria demonstrating their potential use in diagnostic assays.