In this study, copper oxide (CuO) nanoparticles (NPs) are synthesized by the sol-gel method and characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), scanning electron microscopy (SEM) and tunneling electron microscopy (TEM) that all confirmed monoclinic crystalline structure with the particle size of similar to 24 nm. Then, different amounts of doxepin were loaded on the prepared NPs and loading efficacy was evaluated by the linear absorption spectroscopy and nonlinear optical measurements. In the DLS analysis, it is found that enhancing drug loading increases the hydrodynamic radius due to aggregation of NPs and formation of the drug corona around NPs, leading to the increase of light absorption. Moreover, increment of the particle absorption leads to enhancing population of the electrons in the upper level (conduction band) compared to the lower one (valence band) and promotes saturation of the absorption, as confirmed by the open-aperture Z-scan experiment. Finally, to describe the in vitro cellular cytotoxicity of the prepared CuO NPs, this nanomaterial was treated with PC3 human prostate cancer cells and the average growth of cells was monitored by a preliminary MTT assay. This investigation proved a noticeable decrease in the MTT signal compared to the untreated cells; therefore, CuO affects the metabolic activity of the performed cell line. This study shows that CuO can be used as a suitable carrier for a range of drugs, treating PC3 human prostate cancer cells.