Heterogeneous photocatalysts have both potential and demonstrated applications for use in the water-splitting reaction that produces hydrogen, the degradation of organic pollutants, the surface wettability conversion, etc. In this feature article, we have focused on the in-site observation of various reactive oxygen species (ROS), Such as singlet oxygen (O-1(2)) and the hydroxyl radical ((OH)-O-center dot), generated by the photoexcitation of TiO2 nanomaterials using single-molecule fluorescence spectroscopy. The spatially resolved Photoluminescence (PL) imaging techniques enable us to determine the location of the (photo)catalytically active sites that are related to the heterogeneously distributed defects on the surface. We also present the results that revealed the formation and reaction dynamics of the photogenerated charge carriers in individual TiO2 nanoparticles. Furthermore, we introduce the single-molecule single-mismatch detection of the nucleotide sequence upon the photoexcitation of a novel nanoconjugate consisting of TiO2 and DNA on the basis of the mechanistic aspects. Notably, the present conjugates can recognize the difference in a single nucleotide. Consequently, this article provides a significant opportunity to understand the temporal and spatial distributions of ROS generated during the photoirradiation of TiO2 nanomaterials and directly explore the microscopic world in many fields ranging from fundamental physics and chemistry to practical applications.