The significant use of copper oxide nanoparticles (CuO NPs) has generated worries over their impacts on the ecosystem and human health due to their release from numerous products to the environment. The Solanum nigrum L. is recognized as a phytoremediation plant and may survive within the excessive metal-stressed surroundings. Five CuO NPs levels were evaluated for their impacts on the callus of S. nigrum. Fresh, dry weight, water content, free amino acids, and potassium content of callus cells were significantly decreased due to the impact of CuO NPs. We also observed increased levels of malondialdehyde, bound phenolic compounds, soluble carbohydrates and enzymatic activity of peroxidase, and polyphenol oxidase in callus cells supplying conditions for the lowering of oxidative stress triggered by CuO NPs. Phenylalanine ammonia lyase, soluble proteins and free phenolic compounds in callus cells were increased under 50, 100 and 150 mg/L CuO NPs and were significantly decreased in most cases by the application of the highest concentration (200 mg/L) of CuO NPs. The catalase activity in calli didn’t clearly change via CuO NPs stress. Further, Cu accumulation in the callus was increased with increasing levels of CuO NPs in the medium (50–200 mg/L), as evidenced through 10.3, 17.0, 20.9 and 40.4-fold, respectively, as compared with the control. The FT-IR analysis showed alterations in most macromolecules such as phenolic compounds, lipids, proteins, carbohydrates, cellulose, and hemicellulose in callus cells-treated with CuO NPs. From these results, we can conclude that S. nigrum plants can be used to remediate the medium contaminated with CuO NPs because the plant can accumulate the metal and has a response to defend itself from the metal stress.