Rapid growth of nanotechnology has enabled the production and use of engineered nanoparticles (ENPs) in several industries as well as in agriculture areas. This has raised ecotoxicological concerns due to the release of ENPs to the environment. In the present study, we investigated the effects of interactions of copper oxide nanoparticles (CuO NPs) on physiological, biochemical, and molecular indices in seedlings of an important oil seed crop Brassica napus L. The seedlings were treated with 0, 20, 50, 100, 200, 400, and 500 mg/L of CuO NPs for 14 days in halfstrength semi-solid Murashige and Skoog medium. The CuO NPs treatment significantly reduced shoot and root growth as well as plant biomass. Shortening and thickening of primary and lateral roots and inhibition of lateral root growth was observed at higher concentrations. An increase in reactive oxygen species generation, and malondialdehyde accumulation was observed. Histochemical staining of roots with propidium iodide and aniline blue indicated cell death and callose formation in roots. Transcriptional modulation of genes related to oxidative stress viz. CuZn superoxide dismutase, catalase, and ascorbate peroxidase was observed. Element content analysis showed an increase in Cu content and decrease in Fe, Mn, and Zn contents. Overall, exposure to CuO NPs caused oxidative injury, cell death, callose formation, and decreased the micro nutrient contents in B. napus seedlings.