Polymer functionalized metal oxide nanocomposites are great interest due to wide range of application, especially in nanomedicine. The present study reports an eco-friendly bio-inspired synthesis of chitosan/copper oxide (CS-CuO) nanocomposite for the first time using rutin. The bio-synthesized CS-CuO nanocomposite was characterized using UV-Visible spectroscopy, FE-SEM, EDS, TEM, XRD and FTIR analyses. FE-SEM and TEM images revealed the synthesized CS-CuO nanocomposite having colloidal spherical shaped structure with an average size of 10-30 nm. EDS analysis confirmed the elements present in synthesized CS-CuO nanocomposite. FTIR studies revealed the role of rutin and chitosan for reduction, capping and synthesis of CS-CuO nanocomposite from the precursor copper salt. The XRD analysis revealed monoclinic structure of CS-CuO nanocomposite. Anti-proliferative activity of the CS-CuO nanocomposite was evaluated in human lung cancer cells A549. Synthesized CS-CuO nanocomposite showed concentration-depended anti-proliferative activity against A549 cancer cells and their IC value was found to be 20 ± 0.50 μg/mL. Furthermore, synthesized nanocomposite induce apoptosis in treated A549 cancer cells assayed by AO/EtBr fluorescent staining method. In conclusion, the synthesized CS-CuO nanocomposite using rutin can be used as a potential anticancer agent in biomedical and clinical sectors.