<p>Foodborne pathogenic bacteria can cause food poisoning or infection, leading to disease. Early screening of foodborne pathogens has become crucial for managing and preventing these diseases. Here, we developed an electrochemical aptamer-based biosensor to detect <i>Escherichia coli</i> (<i>E. coli O157:H7</i>) in real samples. The glassy carbon electrode (GCE) underwent an initial treatment using a nanocomposite made of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs) (AuNPs/rGO/GCE) to enhance the surface area and boost the sensor’s sensitivity. In addition to good electrical properties, nanocomposites made of rGO and AuNPs play a crucial role in sensor development by enabling high loading and adequate immobilization of biorecognition elements like aptamers while preserving their activity and affinity. Next, to elevate the specificity of the altered electrode, aptamers were linked to the exterior of the prepared electrode (APT/AuNPs/rGO/GCE). The prepared electrode underwent characterization using field emission scanning electron microscopy, energy-dispersive spectroscopy, Fourier-transform infrared spectroscopy, and electrochemical impedance spectroscopy. This proposed aptasensor can accurately identify <i>E. coli O157:H7</i> within a range of 6 to 6 × 10⁸ CFU/mL and has a detection limit of 3.25 CFU/mL. Microbial culture results validate the aptasensor findings, providing quicker results and a lower detection limit.</p>