Folic acid (FA) is used as target drug delivery for cancer cells by adding to copper/chitosan nanocomposite (Cu/CNC) to form FA-Cu-CS complex. Chitosan is a promising polymeric that has received a lot of attention recently. Chitosan nanoparticles have wide applications as a nanocarrier for different organic and inorganic substances. In the present study, chitosan nanoparticles (CNP), Cu/CNC, and FA-Cu-CS had been prepared and characterized. Structural, morphological, optical and anticancer activities of the nanoparticles were studied. X-ray diffraction study showed the formation of CNP, Cu/CNC, and FA-Cu-CS. FE-SEM showed the CNPs have spherical shape. While, FE-SEM image of Cu/CNC has aggregated and semi-spherical shapes, but the image of FA-Cu-CS showed aggregated, agglomerated, and irregular shapes. FTIR analysis resulted peak at 1026.13 cm -1 refer to the CNPs bonded with folic acid. UV/VIS spectroscopy showed absorbance peak at 530 nm due to presence of FA-Cu-CS. Moreover, anticancer activity of CNPs, Cu/CNC and FA-Cu-CS was tested by MTT assay against fibroblast, MCF-7 and ovarian cancer, then it was stained by DAPI to show the morphology of nucleus. FA-Cu-CS has higher cytotoxicity from other nanoparticles which was 54%, 65% and 83% against fibroblast, MCF-7 and ovarian cancer, respectively at concentration of 0.175 μL/mL. To our knowledge, there is no report on enhancing the anticancer activity of copper/chitosan nanocomposite by conjugated with folic acid and comparison of anticancer activity among CNPs, Cu/CNC and FA-Cu-CS.