Copper oxide nanofibers (CuO-NFs) prepared by electrospinning and subsequent thermal treatment processes were demonstrated for the first time for glucose non-enzymatic determination. The structures and morphologies of CuO-NFs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray diffraction spectrum (XRD). Different dispersants were utilized for the suspension preparation and effects of ultrasonic time on the films electrode fabrication were investigated in detail. The assay performances to glucose were evaluated by cyclic voltammetry (CV) and chronoamperometry (I-t). Results revealed a high sensitivity (431.3 microAmM(-1)cm(-2)), fast response (about 1s), long-term stability and excellent resistance towards electrode fouling in the glucose determination at +0.40V. The improved performances of CuO-NFs films electrode for electro-oxidation glucose were ascribed to the high surface-to-volume ratio, complex pore structure, extremely long length of the as-prepared CuO-NFs, and the excellent three-dimensional network structure after immobilization. Results in this study suggest that electrospun CuO-NFs is a promising 1-D nanomaterial for further design and microfabrication of bioelectrochemical nanodevices for glucose determination.