The present study reports an environmentally safe synthesis of CuO NPs employing Echinophora platyloba DC extract. The production of CuO NPs was authenticated using various analytical characterization techniques such as FT-IR, TEM, XRD, SEM-EDX, DLS, zeta potential, and UV-Vis. The DPPH assay was employed to assess the scavenging efficacy of synthesized CuO NPs. The results of the MTT assay exhibited that CuO NPs have efficient anticancer potential on two used cancer cell lines (MCF-7 and Burkitt's lymphoma). Thus, it tempts us to study the synthesized CuO NPs biocompatible aspects by interactions with several biologically significant molecules. The present experimental evidence demonstrates the direct interaction of CuO NPs with DNA, which might be one of the credible highways elevating its anticancer potential. Moreover, we investigated the interaction of CuO NPs with the most important blood proteins (HSA, HHb, and Lys) in simulated physiological conditions using spectroscopic methods. The interaction findings suggest that the native conformation of the above proteins was preserved at the level of secondary structure in spite of complex formations with CuO NPs. The binding constant values revealed the greater binding affinity of CuO NPs with HSA, HHb, and Lys as compared to ct-DNA; the binding affinity was found in the order HSA > HHb > Lys > ct-DNA.