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A novel whole-cell electrochemical sensor was developed and applied for sensitive amperometric detection of riboflavin. In this work, a whole-cell based riboflavin redox cycling system was characterized, in which electroactive bacteria Shewanella oneidensis MR-1 was employed as the biocatalyst to regenerate the reduced riboflavin after the electrode oxidation. This redox cycling system efficiently enhanced the electrochemical response of riboflavin and enabled a stable current output at poised electrode potential. Thus, a sensitive amperometric biosensing system for riboflavin detection was developed by integrating this whole-cell redox cycling system with the conventional riboflavin electrochemical sensor. Remarkably, this riboflavin biosensor exhibited high sensitivity (LOD = 0.85 ± 0.09 nM, S/N = 3), excellent selectivity and stability. Additionally, reliable analysis of real samples (food and pharmaceutical samples) by this biosensor was achieved. This work provided sensitive and practical tool for riboflavin detection, and demonstrated that the integration of electroactive bacteria and using its outwards electron transfer for redox cycling would be a powerful and promising strategy to improve the performance of electrochemical sensing system. [ABSTRACT FROM AUTHOR]