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The increasing global water pollution leads to the need for urgent development of rapid and accurate water quality monitoring methods. Microbial fuel cells (MFCs) have emerged as real-time biosensors for biochemical oxygen demand (BOD), but they grapple with several challenges, including issues related to reproducibility, operational stability, and cost-effectiveness. These challenges are substantially shaped by the selection of an appropriate air-breathing cathode. Previous studies indicated a critical influence of the cathode on both the enduring electrochemical performance of MFCs and the taxonomic diversity at the electroactive anode. However, the effect of different gas diffusion electrodes (GDE) on 3D-printed single-chamber MFCs for BOD biosensing application and its effect on the bioelectroactive anode was not investigated before. Our study focuses on comparing GDE cathode materials to enhance MFC performance for precise and rapid BOD analysis in wastewater. We examined for over 120 days two Pt-coated air-breathing cathodes with distinct carbonaceous gas diffusion layers (GDLs) and catalyst layers (CLs): cost-effective carbon paper (CP) with hand-coated CL and more expensive woven carbon cloth (CC) with CL pre-applied by the supplier. The results show significant differences in electrochemical characteristics and anodic biofilm composition between MFCs with CP and CC GDE cathodes. CP-MFCs exhibited lower sensitivity (16.6 C L mg−1 m−2) and a narrower dynamic range (25 to 600 mg L−1), attributed to biofouling-related degradation of the GDE. In contrast, CC-MFCs demonstrated superior performance with higher sensitivity (37.6 C L mg−1 m−2) and a broader dynamic range (25 to 800 mg L−1). In conclusion, our study underscores the pivotal role of cathode selection in 3D-printed MFC biosensors, influencing anodic biofilm enrichment time and overall BOD assessment performance. We recommend the use of cost-effective CP GDL with hand-coated CL for short-term MFC biosensor applications, while advocating for CC GDL supplied with CL as the preferred choice for long-term sensing implementations with enduring reliability. [ABSTRACT FROM AUTHOR]