An effective tool for early-stage selective detection of the foodborne bacterial pathogen Shigella flexneri (S. flexneri) is essential for diagnosing infectious diseases and controlling outbreaks. Here, a label-free electrochemical DNA biosensor for monitoring S. flexneri is developed. To fabricate the biosensor, detection probe (capture probe) is immobilized on the surface of poly melamine (P-Mel) and poly glutamic acid (PGA), and disuccinimidyl suberate (DSS) functionalized flexible indium tin oxide (ITO) electrode. Anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) is used as a signal indicator for the detection of S. flexneri. The proposed DNA biosensor exhibits a wide dynamic range with concentration of the targets ranging from 1 × 10−6 to 1 × 10−21 molL−1 with a limit of detection (LOD) of 7.4 × 10−22 molL−1 in the complementary linear target of S. flexneri, and a detection range of 8 × 1010–80 cells/ml with a LOD of 10 cells/ml in real S. flexneri sample. The proposed flexible biosensor provides high specificity for the detection of S. flexneri compared to other target signals such as discrete base mismatches and different bacterial species. The developed biosensor displayed excellent recoveries in detecting S. flexneri in spiked food samples. Therefore, the proposed biosensor can serve as a model methodology for the detection of other pathogens in a broad span of industries.