Nonthermal plasma, a nondestructive, fast, and highly reproducible surface functionalization technique, was used to introduce desired functional groups onto the surface of carbon powder. The primary benefit is that it is highly scalable, with a high throughput, making it easily adaptable to bulk production. The plasma functionalized carbon powder was later used to create highly specific and low-cost electrochemical biosensors. The functional groups on the carbon surface were confirmed using NH3-temperature-programmed desorption (TPD) and X-ray photoelectron spectroscopy (XPS) analysis. In addition, for biosensing applications, a novel, cost-effective, robust, and scalable electrochemical sensor platform comprising in-house-fabricated carbon paste electrodes and a miniaturized E-cell was developed. Biotin-Streptavidin was chosen as a model ligand-analyte combination to demonstrate its applicability toward biosensor application, and then, the specific identification of the target Escherchia coli O157:H7 was accomplished using an anti-E. coli O157:H7 antibody-modified electrode. The proposed biosensing platform detected E. coli O157:H7 in a broad linear range of (1 × 10-1-1 × 106) CFU/mL, with a limit of detection (LOD) of 0.1 CFU/mL. In addition, the developed plasma functionalized carbon paste electrodes demonstrated high specificity for the target E. coli O157:H7 spiked in pond water, making them ideal for real-time bacterial detection. © 2022 American Chemical Society. All rights reserved.