 In the present study, multi-spectroscopic methods were applied to investigate the effect of NiO nanoparticles on the structure, activity, and stability of proteinase K. To this aim, UV–Vis spectroscopy, circular dichroism photometry, and spectrofluorometry were conducted. UV absorption spectroscopy results revealed that with the addition of NiO nanoparticles, the maximum absorption of the enzyme showed hyperchromism. The regular decrement in the intrinsic fluorescence intensity of the enzyme was obtained with the increase in NiO nanoparticles concentrations. Far-circular dichroism also demonstrated a reduction in the β-sheet and β-turn and an increase in the α-helix content. Furthermore, the results obtained from this investigation revealed that the catalytic activity and thermal unfolding of the enzyme were improved in the presence of NiO nanoparticles in a concentration-dependent manner. Thus, NiO nanoparticles acted as an activator and stabilizer of proteinase K. Finally, the thermodynamic parameters showed that the enzyme–NiO nanoparticles combination was spontaneous and that the hydrophobic interactions and electrostatic forces played a key role in the interaction of the nanoparticles with the enzyme.