Three-dimensional (3D) sea-urchin-like hierarchical TiO2 microspheres were synthesized by a template-free hydrothermal method. The effects of preparation parameters on the microstructure of 3D sea-urchin-like hierarchical TiO2 were investigated using scanning electron microscopy (SEM), transmission electron microscopy, X-ray diffractometer, energy-dispersive X-ray spectrometer and Brunauer–Emmett–Teller technologies. The growth mechanism and photocatalytic activity of 3D sea-urchin-like TiO2 microspheres were discussed. The results of electron microscopy characterizations SEM showed that the microspheres were consisted of numerous one-dimensional (1D) nanorods. A three-step growth model: oxygenated to be 1D nanorods, self-assembly and protonation, was proposed to illustrate the growth mechanism of sea-urchin-like structures. The synthesized 3D sea-urchin-like hierarchical TiO2 microspheres exhibited a better photocatalytic activity for photodegradation of rhodamine B under sunlight irradiation compared to that of P25, which was attributed to the special 3D hierarchical nanostructure, the increased number of surface active sites and anatase crystal structure.