AbstractDetection of pathogenic bacteria requires fast, reliable, and robust devices while testing food and water samples owing to the potentially lethal effect they have on humans. There has been an exponential rise in the demand for nanotechnology-based sensing platforms which can detect pathogens with utmost specificity. This paper reports the fabrication of a rapid, sensitive graphene oxide-based biosensor employing a direct method for electrochemical detection ofEscherichia coli(E. coli, MTCC 443) bacteria in aqueous samples. Graphene oxide (GO) is considered as one of the most promising materials for future biosensors due to its well-known electrical, physical, and optical performance. In addition, it acts as a sensing element due to its biocompatibility againstE. colileading to a change in conductance which can be measured using cyclic voltammetry and amperometry. The results showed that the sensor gives an optimum response at 37 °C with apHof 7.5 and linearity ranging from 10−1to 10−5cfu mL−1with a detection limit of 100 cfu mL−1. The results were compared with the known conventional methods of bacterial measurements such as plating onto agar medium and UV–visible spectroscopy and gave a correlation coefficient of 0.92. The sensor would not require any tedious pretreatment and is suitable for screening as well as for detection purposes as a result of its ease of use, fast response time, sensitivity, and selectivity.