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.