TiO2 nanofibers (TNFs) with different anatase/rutile phase ratios were fabricated using electrospinning technique followed by the annealing at different temperatures. The effect of annealing temperatures on their morphology, structural, and optical properties and photocatalytic activity was investigated. The photocatalytic performance of TNFs was evaluated by degradation of methyl orange (MO) in aqueous solution under the irradiation of simulated solar light. Annealing temperature significantly influenced photocatalytic degradation of MO due to the incorporation of rutile phase which suppresses recombination of photoactivated electron and hole pairs. Turnover frequency (TOF) of MO degradation was introduced to describe the intrinsic activity of TNFs. TNFs acquired best anatase/rutile phase ratio (A/R = 83/17) when annealed at 650 °C, resulting in highest TOF value 2394 h−1, two times higher as compared to P25 with similar anatase/rutile phase ratio (A/R = 85/15). Appropriate crystalline structure could be the reason for good photocatalytic activity as well as intrinsic activity of TNFs.