© 2015 Sociedade Brasileira de Quimica.1-D nanostructures are promising materials for development of electrochemical devices offering benefits such as fast electron transfer rates and large surface areas. Copper oxide nanofibers (CuO-NFs) synthesized by electrospinning technique and subsequent thermal treatment, were used to modify paraffin-impregnated graphite electrode (PIGE) for a sensitive non-enzymatic glucose detection. The structure and morphology of CuO-NFs were characterized by scanning electron microscopy and transmission electron microscopy. The electrocatalytic activity towards glucose oxidation was evaluated by cyclic voltammetry and chronoamperometry. The results reveal a wide linear response to glucose ranging from 1.0 × 10-6 to 1.93 × 10-3 mol L-1 (R2 = 0.9927). The limit of detection was 0.39 × 10-6 mol L-1 (LOD = 3δ/s). The high aspect ratio of the nanofibers arranged in a three-dimensional network structure significantly enhances the electron transfer process. The electrode preparation is simple and rapid execution, and more importantly the graphite rod is relative low-cost and easy to achieve surface renewal for reusability.