TiO2 hierarchical microspheres assembled by nanotubes (TiO2NTMS) were synthesized via hydrothermal method, acid-washing procedure and subsequent calcination. The morphology, crystal structure and surface area of TiO2NTMS were characterized. The photocatalytic activity was evaluated by degradation of methyl orange as a model molecule under UV irradiation. Experimental results revealed that the crystallinity was improved with increasing the calcination temperature, but specific surface areas and pore volumes steadily decreased. TiO2NTMS calcined at 450°C exhibited the distinctive photocatalytic activity among the calcined catalysts, which was attributed to its good crystallization and remaining tubular structure hierarchical microspheres.