Lithium-doped NiO nanofibers were synthesized using electrospinning followed by calcinations. The morphology, crystal structure, and electrochemical activities of these nanofibers were characterized, respectively, with scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and electrochemical techniques. Electrocatalytic oxidation of glucose was found on these nanofibers in 0.1M NaOH. The catalytic oxidation current was linear with the concentration of glucose in the range of 0.5 to 278μM. The detection limit was calculated to be 0.1μM. Lithium-doped NiO nanofiber is thus a potential electrode material for the construction of non-enzymatic glucose sensor.