This study aimed to develop novel primer-probe sets specifically optimized for detecting E. coli and E. coli O157 through comparative genomic analysis. Genomic data from 2396 E. coli strains, including 228 strains of E. coli O157, were retrieved from the National Center for Biotechnology Information (NCBI). From the analysis, yegR and GDP-mannose mannosyl hydrolase (GDPMH) genes were selected as primer targets for E. coli and E. coli O157:H7, respectively. The sensitivity and specificity of the designed primer-probe sets were evaluated in 48 E. coli strains (including 30 E. coli O157) and 17 non-E. coli strains, totaling 65 strains, and the results indicated high sensitivity and specificity. Tryptic soy broth (TSB), carrots, chicken, milk, potatoes, apple juice, and orange juice did not significantly interfere with detecting E. coli and E. coli O157; however, 50% potato samples did. The efficacy of the designed primers was compared among ground pork, lettuce, and potato samples. Reduced detection efficiency was noted in the lettuce and potato samples. Wavelength interference with components of the lettuce homogenate may have reduced detection efficiency. For potato samples, the high affinity of chlorogenic acid, a compound found in potatoes, for Taq DNA polymerase, a component of the mastermix, contributed to the reduced detection efficiency. The primer and probe sets described in this study, coupled with an understanding of possible interference from food components, will aid in detecting E. coli O157 in the food industry.