In this study, a rapid diagnosis platform was developed for the detection of Escherichia coli O157:H7. An electrical double layer (EDL)-gated field-effect transistor-based biosensor (BioFET) as a point-of-care testing device is demonstrated with its high sensitivity, portability, high selectivity, quick response, and ease of use. The specially designed ssDNA probe was immobilized on the extended gate electrode to bind the target complementary DNA segment of E. coli, resulting in a sharp drain current change within minutes. The limit of detection for target DNA is validated to a concentration of 1 fM in buffer solution and serum. Meanwhile, the results of a Kelvin probe force microscope were shown to have reduced surface potential of the DNA immobilized sensors before and after the cDNA detection, which is consistent with the decreased drain current of the BioFET. A 1.2 kb E. coli duplex DNA synthesized in plasmid was sonicated and detected in serum samples with the sensor array. Gel electrophoresis was used to confirm the efficiency of sonication by elucidating the length of DNA. Those results show that the EDL-gated BioFET system is a promising platform for rapid identification of pathogens for future clinical needs.