Escherichia coli represents a significant concern for food safety, water quality, medical applications, and environmental health, necessitating effective detection methods. This study presents a reduced graphene oxide-based biosensor for the sensitive and specific detection of E. coli DNA. The functionalized reduced graphene oxide was successfully synthesized using a modified Hummers' method, followed by a hydrothermal reduction step. Structural, morphological, and chemical properties of the reduced graphene oxide were confirmed through Fourier-transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. The biosensor incorporates functionalized reduced graphene oxide linked to amino-modified probe DNA sequences specific to E. coli markers, providing stability and selective hybridization. Detection of E. coli DNA in the range of 0-476.19 fM is achieved by measuring absorbance changes at 273 nm, where an increase in absorbance indicates the presence of complementary E. coli single-stranded DNA. Performance testing with various DNA concentrations revealed a linear relationship, achieving a limit of detection of 80.28 fM and demonstrating high selectivity against non-target bacteria such as Bacillus subtilis, Enterococcus, Vibrio proteolyticus, and Staphylococcus. Optimization efforts identified the amino-E.coli BL21 probe as the only probe capable of detecting E. coli DNA. The reduced graphene oxide-based biosensor holds promise for rapid detection in pathogen monitoring, clinical diagnostics, and environmental analysis, paving the way for enhanced DNA biosensing technologies.