Shiga toxin-producing Escherichia coli (STEC) are the leading pathogenic strain causes of diarrhea diseases and death of humans in worldwide. Many diagnostic assays have been developed to aid for the diagnosis of STEC strain; however, they have different limitations. Thus, this study was aimed at designing rapid, effective, sensitive and specific immunodiagnostic assay for STEC strain detection. Thus, a STEC isolate available was recovered from freezer in EMB agar; then a pure typical growth was inoculated into TSB overnight at 37oC. The broth culture was processed using chloroform methanol extraction method for purification of LPS from the bacteria. Assessment on SDS-PAGE showed that the LPS preparation was contaminated by small amount and identity of cellular proteins. The produced antibody showed positive agglutination both on the purified LPS as well as the STEC isolate carrying LPS on their surface; however, agglutination of STEC was more pronounced. Mice immunized with LPS produced highest agglutination on tertiary immunization showing the progressive buildup of the antibody response against the antigen. Cultures from TSA and when they refresh showed better agglutination than cultures on EMB as well as TSB. Immunodiagnostic assay developed in this study could detect STEC strains including STEC in human feces with rapidly (1–2 minutes), sensitively (90.2%), specifically (89.5%) and accurately (90.6%). However, further studies are still required to improve it sensitivity, specificity and reproducibility. Overall this diagnostic assay provided promising results that may curb current problem with detection methods in clinical health care and research laboratories.