Escherichia coli is frequently associated with direct or indirect contact with ruminant faeces. Lauryl tryptose broth with MUG medium tubes inoculated with polluted surface water samples showing gas production with growth is considered a positive indication of E.coli presence . The loopfull of unique growth colony was streaked on MacConkey agar plates ; The culture shows pink to red color surrounded by red zone . While on EMB agar medium given pink growth golden metallic sheen . Three common isolates were identified according Bergey's Manual of Systemic bacteriology as E.coli which gave different positive results. The antigen of E.coli cells were prepared in saline phosphate buffer and emulsified with Freunds complete adjuvant for the period immunization by intramuscular and intravenous injection . Immunization of rabbit with highly purified antigen induced high titer 1:2048 and specificity by indirect ELISA. The serological relationship among three E.coli isolates was clear using obtained antiserum by double diffusion test.