Detection of E. coli in water and food constitutes a major challenge. However, the outer membrane proteins of this Gram-negative bacterium represent an excellent choice for their detection and diagnosis. Two outer membrane proteins: A and C, play important roles as drug modulators and in cellular permeability for this bacterium. Here we employed the antibodies raised against these two outer membrane proteins for the development of dot-blot immunoassays to detect this bacterium in water and meat samples. This immune-based assay was not only sensitive and reliable but rapid and cheap to perform. Sensitivities and specificities were demonstrated at the lowest detection limits of just two to three cells per milliliter, while the upper limit reached 107 cells per milliliter. In conclusion, the simplicity and extremely high resolution of this colored-based immunoassay test would be of great importance for untrained food and/or water inspectors or laboratory personnel to use with immediate visual results.