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Detection of bacterial pathogens is significant in the fields of food safety, medicine, and public health, just to name a few. If bacterial pathogens are not properly identified and treated promptly, they can lead to morbidity and mortality, also possibly contribute to antimicrobial resistance. Current bacterial detection methodologies rely solely on laboratory-based techniques, which are limited by long turnaround detection times, expensive costs, and risks of inadequate accuracy; also, the work requires trained specialists. Here, we describe a cost-effective and portable 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5α, BL21, TOP10, and JM109) within 15 min using 500 μL of sample, and costs only USD 2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 cfu, limit of quantification (LOQ) of 270 cfu, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a strong candidate for frequent testing at point of care; it also has application in various fields and industries where pathogen detection is of interest.