Monodisperse TiO2 nanoparticles and urchin-like hierarchical TiO2 nanospheres assembled with ultrathin quantum nanowires (about 2 nm) have been synthesized by a simple template-free wet chemical method. The morphology, structure, and crystallinity of the TiO2 nanomaterials were investigated by field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), and high resolution transmission electron microscopy (HRTEM). Electrochemical measurements with the hierarchically nanostructured TiO2 nanospheres as an electrode showed much better reversibility for direct electrochemistry of cytochrome c (cyt c) and much higher sensitivity than for an electrode composed of the monodisperse TiO2 nanoparticles. The excellent performance of the hierarchical TiO2 nanospheres may result from a quantum size effect, and their favorable nanostructure (with the presence of an abundance of both uniform macropores and mesopores), excellent structural stability and high specific surface area. The relative ionic strength had significant effect on the direct electrochemistry. Very high ionic strengths relative to cyt c concentration (I/c) induced a conformational change of cyt c on the nanostructure-coated electrode, from the native state to a partially unfolded one in 25 mmol/L phosphate buffer solution (pH 6.8).