Shuttle-like copper oxide (CuO) was prepared by a hydrothermal decomposition process. The resulting material was characterized by scanning electron microscopy and X-ray diffraction. It was then immobilized on the surface of a glassy carbon electrode modified with a film of poly(thionine). A pair of well-defined and reversible redox peaks for Hg(II) was observed with the resulting electrode in pH 7.0 solutions. The anodic and cathodic peak potentials occurred at 0.260 V and 0.220 V (vs. Ag/AgCl), respectively. The modified electrode displayed excellent amperometric response to Hg(II), with a linear range from 40 nM to 5.0 mM and a detection limit of 8.5 nM at a signal-to-noise ratio of 3. The sensor exhibited high selectivity and reproducibility and was successfully applied to the determination of Hg(II) in water samples.