Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study,Escherichia coliRosetta was genetically modified to expressOprFandribBwith promoter P(t7)and P-cusC, respectively, which could synthesize porin and senses Cu(2+)to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45-3.56 mu M) was positively correlated to Cu2+(0-0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37 degrees C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu(2+)concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively;R-2 = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu(2+)with calculated detection limit of 28 mu M, which conforms to the common Cu(2+)safety standard (32 mu M). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC-based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu(2+)in water.