Chitosan/CuO nanocomposites (Chi/CuO) were prepared by facile and eco-friendly technique. The 2%w/v chitosan solution was mixed with 0.5 %w/w sodium tripolyphosphate (STPP), resulting in the formation of ionically crosslinked chitosan. The crosslinked chitosan was soaked in an aqueous solution containing 0.001, 0.01 or 0.1 mol/L CuSO4·5H2O for 24 hrs, in which the Cu2+ ions were absorbed into the chitosan network, forming as the chitosan/Cu2+ precursors. The chitosan/Cu2+ precursors were hydrothermally reacted in two different basic media, i.e. NaOH and NH4OH, at 100°C for 24 hrs, resulting in the nano-sized CuO crystals hydrothermally grew and embedded in the crosslinked chitosan matrix. The CuO grown in the NaOH possessed larger crystallite size and higher crystallinity than that in the NH4OH. In addition, the CuO crystallite size in the nanocomposites increased with the increase of initial concentration of Cu2+ starting agent due to the increase of Cu2+ quantity in the chitosan/Cu2+ precursors. The chitosan/CuO nanocomposites prepared by using 0.01 and 0.1 mol/L Cu2+ could exhibit the antibacterial activities after intimate contact with Staphylococcus aureus and Escherichia coli under JIS L 1902:1998 (Qualitative) test method, indicating their potential use as biocontrol agents.