Detecting microorganisms quickly and selectively is extremely important in clinical analysis and in monitoring the quality of food and water. This study details the development of a biosensor that uses electrochemical methods to selectively detect uropathogenic Escherichia coli (E. coli) bacteria in both aqueous and serum samples. The biosensor was developed using a simple and cost-effective method involving reduced graphene oxide (r-GO) with PVA (Polyvinylalcohol) and PEI (Polyethylenimine) through a Sol-Gel spin coating process. The numerous NH2 groups on the PVA and PEI were used to functionalize the biosensor's surface. To increase the specificity of the detection process, amide bonds were formed on the electrode surface using anti-fimbrial E. coli antibodies. The redox mediator prevents the formation of immunological complex and it enhances the transmission of electrons from the developed PVA/rGO/PEI-modified layer to detect E. coli. The development of an electrochemical test for Escherichia coli illustrated the efficacy of these biosensors. Using only 5 µL of the sample, it was discovered that these biosensors had a broad dynamic range (915-2.5×107 CFU/mL) and low limits of detection (285 CFU/mL). Moreover, the biosensor performed well in aqueous, serum, and urine media, making it potentially useful for the clinical diagnosis of pathogenic diseases. This study highlights the potential of these biosensors for real-world, point-of-care applications.