Background: Copper oxide nanoparticles(CuO NPs) have been powerful evidence in several in vitro studies such as cytotoxicity and antimicrobial compared with other metal oxide. Here, we have synthesized green CuO NPs using Coix lacryma jobi leaves extracts. Place and Duration of Study: Department of Chemistry  Manipur University, Manipur, India and Regional Institute of Medical Sciences, Imphal, India between February 2019 to March 2021. Methodology: Green CuO NPs nanoparticles were synthesized from Copper chloride dihydrate (CuCl2.2H2O) using Coix lacryma jobi leaves extract, and the synthesized green CuO NPs were characterized using Field Emission Scanning Electron Microscopy (FESEM) - Energy Dispersive Spectroscopy, IR Spectroscopy, UV-Visible Spectroscopy, Powder X-Ray diffraction Spectroscopy and HR-TEM where FESEM-EDS determined the purity of CuO NPs. Results: No other impurities present were observed in EDS, and the PXRD spectra show the crystallite size of CuO NPs with respect to the (002) plane is found to be 25.2 nm, and the presence of CuO NPs at adsorption spectrum with a distinct peak at 282 nm was determined by  UV-Visible spectroscopy and the homogenous morphology and crystalline nature of the CuO NPs were determined from TEM micrograph and SAED pattern. In applications, the substantial anticancer activity of green CuO NPs (synthesized using Coix lacryma jobi leaves extract) was proved on human cervical and lung cancer cell lines with IC50 values of 31.88 μg/ml and 15.61 μg/ml, respectively.