The newly designed preparation route of the crystalline TiO2 nanofibers by electrospinning was described. Relationships between calcination temperature and the precursor amount for the transition of titanium (IV) isopropoxide (Ti(Iso)) to crystalline titanium dioxide (TiO2) were thoroughly studied. The electrospinning solutions were obtained by adding the increasing amount of Ti(Iso) as a precursor, together with acetic acid to the 10 wt% Polyvinylpyrrolidone (PVP)/ethanol solution. The effect of Ti(Iso) concentration on structural and morphological characteristics of PVP/Ti(Iso) and obtained TiO2 nanofibers were investigated by various methods; Attenuated Total Reflectance-Fourier transform infrared spectroscopy-(FTIR-ATR), Raman and XRD spectroscopy, Thermal gravimetric analysis (TGA), Scanning electron microscopy (SEM) and Physical adsorption of nitrogen. SEM image of the calcinated sample showed that resulting TiO2 nanofibers were obtained with a diameter of approximate to 150 nm after calcination at 450 degrees C. Raman measurements evidenced the formation of TiO2 nanofibers in anatase form. It was proved by photo-electrochemical measurements that the resulted TiO2 nanofibers layer really reflects the ability to react to a UV light signal by generating of the charge carriers, which is promising for any subsequent applications.