Molybdenum-doped TiO2 nanotubes (TNTs) samples were synthesized in two routes by a single-step anodization method for a sustainable photocatalytic process. This study aimed to evaluate the effectiveness of dopant adding time on the photocatalytic activity of TiO2 nanotubes. In the first route, molybdenum dopant with three initial concentrations of 0.02, 0.06, and 0.1 M was added to the electrolyte at the beginning of the anodizing process, while in the second route, molybdenum precursors were added to the electrolyte after 30 min from the beginning of anodization (after the formation of nanotubes). The structural characteristics of Mo-doped TNTs in both routes were compared with non-doped nanotubes using field emission scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS), and X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) techniques. The optical properties and efficiency of electron-hole pairs separation and charge transfer kinetics were evaluated by diffuse reflectance spectroscopy (DRS) and electrochemical impedance spectroscopy (EIS) methods. Moreover, methylene blue (MB) degradation under visible light was performed to investigate the photocatalytic activity. The results indicate that the second route of synthesis causes the formation of well-aligned TNTs while increasing the Mo concentration will destroy the tube structure in some places on the surface and cause the loss of structural order, in the first path. By Mo doping into TiO2 nanotubes, the optical properties are improved and the absorption edge of TiO2 nanotubes was extended to the visible region. The Mo-doped samples in the second route with concentrations of 0.1 M exhibited the highest visible light absorption and charge transfer rate among all the Mo-doped samples. Also, Mo-doped samples had better photocatalytic activity than non-doped samples in MB degradation and the highest photocatalytic efficiency was related to the Mo-doped sample synthesized in the second route with a Mo concentration of 0.1 M.