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Chitosan is a linear polysaccharide and non-toxic bioactive polymer with a wide variety of applications due to its functional properties such as ease of modification, and biodegradability. In this study, a green protocol for supporting of Cu(II) on chitosan-encapsulated magnetic Fe3O4 nanoparticles is described. The morphological and physicochemical features of the material were determined using several advanced techniques like fourier transformed infrared spectroscopy (FT-IR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), inductively coupled plasma (ICP), vibrating sample magnetometer (VSM) and X-ray photoelectron spectroscopy (XPS). The average diameter of the NPs was approximately 15–25 nm. In addition, the Fe3O/CS/Cu(II) nanocomposite was engaged in biological assays like study of anti-oxidant properties by DPPH mediated free radical scavenging test using BHT as a reference molecule. Thereafter, on having a significant IC50 value in radical scavenging assay, we extended the bio-application of the desired nanocomposite in anticancer study of lung well-differentiated bronchogenic adenocarcinoma, lung moderately differentiated adenocarcinoma, and lung poorly differentiated adenocarcinoma of human lung in-vitro conditions. In the cytotoxicity and anti-human lung studies, the nanocomposite was treated to lung cancer lung well-differentiated bronchogenic adenocarcinoma (HLC-1), lung moderately differentiated adenocarcinoma (LC-2/ad), and lung poorly differentiated adenocarcinoma (PC-14) cell line following MTT assay. The cell viability of malignant lung cell line reduced dose-dependently in the presence of Fe3O/CS/Cu(II) nanocomposite. The recent results suggest that Fe3O/CS/Cu(II) nanocomposite have a suitable anticancer activity against lung cell lines.