A novel antibacterial thermoplastic polyurethane (TPU) nanofiber membrane was fabricated by electrospinning with the aid of Cu-loaded NaX zeolite (CuX), which showed excellent and long-lasting antibacterial activity against Escherichia coli (E. coli). The crystalline structure, morphology and composition of the obtained NaX zeolite, CuX and CuX/TPU nanofiber membrane were analyzed by X-ray diffraction (XRD), scanning electron microscope (SEM) and X-ray fluorescence (XRF), respectively. The antibacterial activity of CuX/TPU nanofiber membrane against E. coli was investigated by inhibition zone method and plate-counting method, and it is indicated that the inactivation efficiency was increased from 58.40 to 99.85% with increasing the addition of CuX from 5 to 30% in 4h. The result of kinetics study indicated that the disinfection process accorded with the pseudo-first-order model. The possible antibacterial mechanism was also proposed that the released Cu2+ by CuX in nanofiber membrane destroyed the cell wall and penetrated plasma membrane of E. coli. It is expected that this CuX/TPU nanofiber membrane with excellent antibacterial activity would be a promising candidate for biomedical applications.