Toxicological effects of nanoparticles (NPs) are still poorly documented while there are great demands for industrial applications and daily life. The aim of this study is to evaluate the influence of physicochemical characteristics on TiO₂ NP toxicological effects toward protein. In order to better understand the physicochemical basis of the toxic of NP in industrial applications and under conditions of environmental exposure, we performed an array of photophysical measurements to quantify the interaction of TiO₂ NP with protein. Fluorescence quenching, circular dichroism, dynamic light scattering and transmission electron microscopy measurements were performed on TiO₂ NP having a diameter range from 10 to 35 nm in the performance of protein. We find that the TiO₂ NP strongly associates with protein where the binding constant, as well as the degree of cooperativity of particle-protein binding, depends on particle size. We also find tentative evidence that the protein undergoes conformational change upon association with the NP. These results indicate that exposure to TiO₂ NP may have an unfavorable effect on human health by inactivating functional proteins.