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Infections caused by microorganisms are a public health problem worldwide. New biodetection systems are essential to diagnose with accuracy resulting in more effective treatment. In this work, we propose a ConA-conjugated graphene quantum dots and polypyrrole film-based biosensor for label-free detection of Candida albicans, Candida glabrata, Candida tropicalis, E. coli, B. subitilis, and S. aureus. We modified polypyrrole and graphene quantum dots (PPY-QDGs) with Concanavalin A (Con A) lectin. ConA is a glucose/mannose-specific lectin. The results showed that ConA lectin has the highest binding affinity for C. tropicalis and S. subtilis. PPY-GQDs-ConA binding profile revealed differential response for Candida spp (C. tropicalis > C. albicans > C. glabrata) and bacterial (B. subtilis > S. aureus > E. coli). The limits of detection (LOD) obtained were 1.42 CFU/mL for C. albicans, and 3.72 CFU/mL for C. glabrata. C. tropicalis yielded a LOD of 0.18 CFU/mL. The respective LODs for the evaluated bacteria were 0.39 CFU/mL for S. aureus, 0.72 CFU/mL for S. subtilis, and 2.63 CFU/mL for E. coli. The differential response obtained for the sensor can be attributed to the heterogeneous distribution of carbohydrates on the microorganism's surfaces. The proposed system based on a flexible substrate is effective for microbiological diagnosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)