In this work, the preparation of copper oxide (CuO) nanosheets by laser ablation in water was demonstrated. The optical, structural, stability, and morphological properties of CuO nanosheets were investigated using UV–Vis spectrophotometer, X-ray diffraction (XRD), scanning electron microscope (SEM), and zeta potential (ZP). The XRD results confirm the formation of crystalline (CuO) with a monoclinic phase. The optical energy gap of CuO nanosheets was found to be 2.2 eV at 300 k. The average thickness of the synthesized copper oxide nanosheets is 25 nm. Energy dispersive X-ray result confirms the formation of stoichiometric CuO. The zeta potential result confirms the synthesized CuO colloid is stable. Gram-positive bacteria are found to be more susceptible to CuO nanosheets than Gram-negative bacteria. The synthesized copper oxidenanosheets are tested for antibacterial activity against Gram (+ve) and Gram (-ve) bacteria stains. The results suggest that CuO NPS can give hydrogen atoms and remove the unstable electron from DPPH at a rate of 200 μg/ml than 12.50 μg/ml. The results demonstrate that the copper oxide nanosheets have an effective antioxidant. Furthermore, according to a cytotoxicity assay, when cancer cells are incubated with copper oxide nanosheets, they are unable to grow well. copper oxide nanosheets can induce apoptosis and suppress pancreatic cell proliferation.