Highly transparent nanocrystalline TiO2 films have been fabricated by electrospinning (ES) technique based on a transmutation process from as-spun nanofibers with an appropriate amount of tri-ethanolamine (TEOA) added to the precursor. A possible evolution mechanism of the transparent nanocrystalline TiO2 films is proposed. It is found that the films prepared via transmutation from electrospun nanofibers possess rich bulk oxygen vacancies (BOVs, PL band at 621–640 nm) by using photoluminescence (PL) spectroscopy. Contrastively, the dominant peak in PL spectrum of the spin-coated film is the emission from surface oxygen vacancies (SOVs, PL band at 537–555 nm). The electrospun TiO2 films with rich BOVs induce large open-circuit voltage (V oc) and fill factor (FF) improvements in dye-sensitized solar cells (DSCs), and thus a large improvement of energy conversion efficiency (η). In addition, these performance advantages are maintained for a double-layer cell with a doctor-bladed ∼7 μm top layer (P25 nanometer TiO2, Degussa) and an electrospun ∼3 μm bottom layer. The double-layer cell yields a high η of 6.01%, which has increased by 14% as compared with that obtained from a 10 μm thick P25 film.