The requirement to prevent foodborne illnesses underscores the need for reliable detection tools, stimulating biosensor technology with practical solutions for in-field applications. This study introduces a low-cost immunosensor based on a single-walled carbon nanotube (SWCNT)-modified gold leaf electrode (GLE) for the sensitive detection of Escherichia coli. The immunosensor is realized with a layerby-layer (LbL) assembly technique, creating an electrostatic bond between positively charged polyethylenimine (PEI) and negatively charged carboxyl-functionalized SWCNTs on the GLE. The structural and functional characterization of the PEI-SWCNT film was performed with Raman spectroscopy, high-resolution scanning electron microscopy (HRSEM), and electrical measurements. The PEI-SWCNT film was used as a substrate for antibody immobilization, and the electrochemical sensing potential was validated using electrochemical impedance spectroscopy (EIS). The results showed a wide dynamic range of E. coli detection, 10 1 −10 8 cfu/mL, with a limit of detection (LOD) of 1.6 cfu/mL in buffer and 15 cfu/mL in the aqueous solution used for cleansing fresh lettuce leaves, affirming its efficiency as a practical and affordable tool in enhancing food safety.