The primary objective of this study is to explore the interaction of β-galactosidase with copper oxide nanoparticles (CuO NPs). Steady-state absorption, fluorescence and circular dichroism (CD) spectroscopic techniques have been employed to unveil the conformational changes of β-galactosidase induced by the binding of CuO NPs. Temperature dependent fluorescence quenching results indicates a static quenching mechanism in the present case. The binding thermodynamic parameters delineate the predominant role of H-bonding and van der Waals forces between β-galactosidase and CuO NPs binding process. The binding was studied by isothermal titration calorimetry (ITC) and the result revealed that the complexation is enthalpy driven, the ΔH°<0, ΔS°<0 indicates the formation of hydrogen bonds between β-galactosidase and CuO NPs occurs. Disruption of the native conformation of the protein upon binding with CuO NPs is reflected through a reduced functionality (in terms of hydrolase activity) of the protein CuO NPs conjugate system in comparison to the native protein and CuO NPs exhibited a competitive mode of inhibition. This also supports the general belief that H-bond formation occurs with NPs is associated with a lesser extent of modification in the native structure. Morphological features and size distributions were investigated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Additionally the considerable increase in the Rh following the addition of CuO NPs accounts for the unfolding of β-galactosidase. Chemical and thermal unfolding of β-galactosidase, when carried out in the presence of CuO NPs, also indicated a small perturbation in the protein structure. These alterations in functional activity of nanoparticle bound β-galactosidase which will have important consequences should be taken into consideration while using nanoparticles for diagnostic and therapeutic purposes.