The present work demonstrates how the growth and photoelectrochemical (PEC) properties of anodic TiO2 nanotubes (NTs) are influenced by Ti substrates in the new HBF4 containing electrolyte. Three different Ti substrates, the rough abraded Ti foil, the smooth electropolished Ti and the two-step Ti plate (i.e. the Ti substrate with ordered dimples left by removing the TiO2 NTs formed in the first-step anodization), are employed for the preparation of TiO2 NTs. It is revealed that the Ti substrate morphology can largely influence the formation of TiO2 NTs, particularly in the initial tube generation stage. The two-step Ti substrate has ordered hexagonally distributed dimples which play the role of template for the formation of new TiO2 NTs. As a result, the TiO2 NTs grown via a two-step anodization approach have highly ordered structure and extremely smooth sidewalls, hence possess the best PEC performance among the three kinds of substrates.