A new anodization based method is developed to fabricate a gradient in TiO2 nanotube diameters and lengths on Ti foil. In the method, the applied anodization voltage is increased step by step, while the Ti foil is immersed into an aqueous solution of hydrofluoric acid progressively during anodization. The gradient TiO2 nanotubes with tube diameters ranging from 55 to 105 nm and lengths ranging from 300 to 500 nm across 12 mm of Ti foil are obtained. The formation mechanism of gradient diameter and length is also discussed. The gradient structure is potentially useful as new cell instructive materials (CIMs) for guided cell movement and culture, novel drug delivery vehicles, size-selective biosensors, or other sensor applications.