A bio-electrochemical strategy was developed for constructing a simple and sensitive levofloxacin (LEV) sensor based on a single chamber microbial fuel cell (SC-MFC) using FePO4 nanoparticles (NPs) as the cathode catalyst instead of traditional Pt/C. In this assembled sensor device, FePO4 NPs dramatically promoted the electrooxidation of oxygen on the cathode, which helps to accelerate the voltage output from SC-MFC and can provide a powerful guarantee for LEV detection. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to fully characterize the FePO4 NPs. Under the optimized COD condition (3 mM), the LEV with a concentration range of 0.1-1000 pg/L could be detected successfully, and exhibited the excellent linear interval in the concentration range of 0.1-100 pg/L. During this range of concentrations of LEV, a temporary effect on the anode of exoelectrogenic bacterial in less than 10 min could occur, and then came back to the normal. It exhibited a long-term stability, maintaining the stable electricity production for 14 months of continuous running. Besides, the detection mechanism was investigated by quantum chemical calculation using density functional theory (DFT).