A whole-cell bioelectrochemical biosensing system for amperometric detection of riboflavin was developed. A "bioelectrochemical wire" (BW) consisting of riboflavin and cytochrome C between Shewanella oneidensis MR-1 and electrode was characterized. Typically, a strong electrochemical response was observed when riboflavin (VB2) was added to reinforce this BW. Impressively, the electrochemical response of riboflavin with this BW was over 200 times higher than that without bacteria. Uniquely, this electron rewiring process enabled the development of a biosensing system for amperometric detection of riboflavin. Remarkably, this amperometric method showed high sensitivity (LOD = 2.2 nM, S/N = 3), wide linear range (5 nM ∼ 10 μM, 3 orders of magnitude), good selectivity, and high resistance to interferences. Additionally, the developed amperometric method featured good stability and reusability. It was further applied for accurate and reliable determination of riboflavin in real conditions including food, pharmaceutical, and clinical samples without pretreatment. Both the cost-effectiveness and robustness make this whole-cell amperometric system ideal for practical applications. This work demonstrated the power of bioelectrochemical signal amplification with exoelectrogen and also provided a new idea for development of versatile whole-cell amperometric biosensors.