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In this paper, the un-doped, Mo doped, N doped and Mo–N co-doped TiO2 nanotubes (TNTs) films were prepared by a two-step anodic oxidation method. The paper mainly investigated the origin of room temperature (RT) magnetic properties of all the samples. X-ray diffraction studies showed that the impurity of magnetic Mo atoms or N atoms were doped into anatase TiO2 lattices. It is revealed by the results of X-ray photoelectron spectroscopy and photoluminescence spectra that oxygen vacancies (Vos) exist. For all the samples the values of the saturation magnetizations (Ms) are in the order of Mo–N co-doped TiO2 > Mo-doped TiO2 > N-doped TiO2 > un-doped TiO2. Thus, the the Mo–N co-doped film achieved the highest RT Ms. The enhancement of the Ms origins from the cooperationg of the doping Vo, Mo and N. This progress provides some clues for the increase the saturation magnetization in TiO2-based dilute magnetic semiconductors at RT. The experiment results indicate that the Vos play a crucial role in RT ferromagnetic properties for TNTs films.