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N-doped TiO₂ with oxygen vacancies exhibits many advantages for photocatalysis, such as enhanced visible light absorbency, inhibition of the photogenerated charge carrier recombination, etc. However, preparation of N-doped TiO₂ with oxygen vacancies under mild conditions is still a challenge. Herein, N-doped TiO₂ nanospheres with tetrahedral Ti⁴⁺ sites were synthesized by using dodecylamine as template and assisted by <span style="font-variant: small-caps;">l</span>-alanine acids. The obtained samples were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV&#8722;Vis diffuse reflectance spectra (UV&#8722;Vis DRS). It was found that the dodecylamine as a neutral surfactant controlled the structure of TiO₂ spherical, while <span style="font-variant: small-caps;">l</span>-alanine acids provided a nitrogen source. The existence of tetrahedral Ti⁴⁺ sites in N-doped TiO₂ was also confirmed. The N-doped TiO₂ sample with tetrahedral Ti⁴⁺ sites exhibited significantly improved photocatalytic performance for degradation of methylene blue solution under UV light or visible light irradiation. A combined time-resolved infrared (IR) spectroscopy study reveals that the enhanced photocatalytic performance could be attributed to a large amount of photogenerated charge carriers and efficient charge separation. It is demonstrated that the shallow donor state produced by oxygen vacancies of tetrahedral Ti⁴⁺ sites can effectively promote separation of charge carriers besides capturing electrons.