The use of TiO2 nanoparticles (NPs) in a wide range of products (specially biomedical applications) in day-to-day life and the notable fact that it is photoactive and generates reactive oxygen species (ROS) create curiousness among researchers to study their effects on different biological systems. Heme-enzymes are known to operate redox reactions in living system in tandem with ROS. Horseradish peroxidase (HRP) being a model heme-enzyme from the peroxidase family was systematically studied for enzymatic changes after interaction with TiO2 NPs under physiologically relevant pH and irradiation conditions. This study gives us an extensive idea of the changes in enzyme kinetics, intrinsic fluorescence, interaction efficiency, and stability of the protein–nanoparticle complex formed. The effect of ROS generated by TiO2 NPs under different irradiation conditions was substantially considered as one of the major factors that influenced the enzyme kinetics apart from protein adsorption on the surface of nanoparticles. The impact of excessive use of TiO2 NPs on the activity of HRP can be discerned from the current study. © 2017 Elsevier B.V.