l-cysteine (l-Cys) detection is of great importance because of its crucial roles in physiological and clinical diagnoses. In this study, a glucose/O-2 biofuel cell (BFC) was assembled by using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH)-based bioanode and laccase-based biocathode. Interestingly, the open circuit potential (OCP) of the BFC could be inhibited by Cu2+ and subsequently activated by l-Cys, by which a BFC-based self-powered sensing platform for the detection of l-Cys was proposed. The FAD-GDH activity can be inhibited by Cu2+ and, in turn, subsequent reversible activation by l-Cys because of the binding preference of l-Cys toward Cu2+ by forming the CuS bond. The preferential interaction between l-Cys and Cu2+ facilitated Cu2+ to remove from the surface of the bioanode, and thus, the OCP of the system could be turned on. Under optimized conditions, the OCP of the BFC was systematically increased upon the addition of the l-Cys. The OCP increment (OCP) was linear with the concentration of l-Cys within 20 nM to 3 mu M. The proposed sensor exhibited lower detection limit of 10 nM l-Cys (S/N = 3), which is significantly lower than those values for other methods reported so far. Other amino acids and glutathione did not affect l-Cys detection. Therefore, this developed approach is sensitive, facile, cost-effective, and environmental-friendly, and could be very promising for the reliable clinically detecting of l-Cys. This work would trigger the interest of developing BFCs based self-powered sensors for practical applications.