Water-borne pathogens are mostly generated due to poor sanitation, industrial effluents, and sewage sludge, leading to a significant increase in mortality rate. To prevent this, we need a simple, user-friendly, and rapid on-site detection tool of pathogens, i.e., a biosensor. As contaminated water mainly contains (80%) coliform bacteria, of which Escherichia coli is the major species, we have developed a screen-printed paper-based, label-free biosensor for the detection of E. coli in water. A nanoarchitectured graphene oxide (GO), as a fast electron-transfer flatland, was deposited on the screenprinted graphene (G) on a hydrophobic paper, followed by the immobilization of lectin Concanavalin A (ConA) as a biorecognition element for a GGO_ConA-biosensing electrode. The electrochemical characterization of GGO_ConA shows fast electron transfer with a calculated electroactive surface area of 0.16 cm 2 . The biosensor performance was tested in the sludge water and beach water (real sample) as an analyte using the electrochemical impedance spectroscopy (EIS) technique. The charge-transfer resistance (R ct ) of GGO_ConA increases linearly with the bacterial concentration in the range of 10−10 8 CFU mL −1 with an estimated limit of detection (LOD) of 10 CFU mL −1 , which indicates the ultrasensitivity of our biosensor, with 100 times more sensitivity than previous studies. Our reported biosensor, being cost-effective, eco-friendly, and ultrasensitive, may serve greatly as a portable monitoring kit for checking water-borne bacterial contamination.