Titanium dioxide (TiO2) photocatalysts have been extensively studied and used for the water-splitting reaction that produces hydrogen, the degradation of organic pollutants, the surface wettability conversion, and so on. It is well-known that electron-hole pairs are generated when TiO2 is irradiated by UV photons with energy higher than the TiO2 band gap energy (3.2 eV) and these charge carriers can migrate to the TiO2 surface to initiate redox reactions with adsorbates. Generation of reactive oxygen species was also detected during the TiO2 photocatalytic reaction. In order to develop new and efficient TiO2 photocatalysts, we have investigated the TiO2 photocatalytic reaction mechanism with time-resolved transient absorption and emission spectroscopies, two-color two-laser flash photolysis and single-molecule fluorescence techniques. Particularly, the single-molecule fluorescence spectroscopy has been demonstrated to overcome the spatial heterogeneities of the nanoscale local environments and the inhomogeneous vibronic coupling between the adsorbed molecules and the rough surfaces of the semiconductors, involved in the interfacial electron-transfer processes.