Aligned nanofibers play a significant role in the organic or inorganic material relevant applications because of their remarkable anisotropy, high surface-to-volume ratio, enhanced mechanical properties and charge transfer efficiency. In this work, highly aligned TiO2 nanofibers were continuously prepared over large areas via an updated electrospinning configuration consisting of two parallel electrodes and one additional assistant electrode. Because of the higher quantity and mechanical strength, the aligned TiO2 nanofibers could form an independent membrane. The morphology, thermal stability, structure, optical properties and charge transfer efficiency of this membrane were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TG), X-ray diffraction (XRD), Raman spectra, X-ray photoelectron spectroscopy (XPS), UV-visible (UV-vis) diffused reflectance spectroscopy (DRS) and electrochemical impedance spectroscopy (EIS). Compared with the traditional electrospun TiO2, the well-aligned TiO2 nanofibrous membrane exhibited excellent photoelectrochemical performance and enhanced photocatalytic degradation efficiency of rhodamine B, which means it is a promising material to replace traditional TiO2 for solar light utilization and organic pollutants degradation.