Mesoporous CuO microspheres with core-in-hollow-shell structure were synthesized by one-pot hydrothermal method, using dicyandiamide as hydrolysis agent and poly(vinylpyrrolidone) as soft template. The as-synthesized CuO microspheres were characterized by powder X-ray diffraction, field-emission scanning electron microscopy and N-2 sorption measurements. The core-in-hollow-shelled CuO microspheres exhibit greatly enhanced amperometric response towards glucose as compared to that of the simple hollow counterpart. The as-developed non-enzymatic glucose sensor exhibited favorable performance with wide linear range (0.005-7.95 mM), sensitivity of 622.2 mu A mM cm(-2) and low detection limit of 1 mu M.