Co(3)O(4) nanofibers were fabricated by a two-step procedure consisting of electrospinning and subsequent calcination. Scanning electron microscopy and transmission electron microscopy were employed to characterize the as-prepared Co(3)O(4) nanofibers. Fourier transform infrared spectroscopy and Raman spectroscopy were used to confirm the degradation of the polymer matrix and the complete conversion of cobalt nitrate to cobalt oxide. Furthermore, the composition and crystal structure of the final product were investigated using X-ray diffraction and X-ray photoelectron spectroscopy. The as-prepared Co(3)O(4) nanofibers were applied to construct a non-enzymatic sensor for glucose detection in alkaline solution. The developed sensor showed a fast response time (less than 7 s), a high sensitivity of 36.25 mu A mM(-1) cm(-2), good reproducibility and selectivity, and a detection limit of 0.97 mu M (S/N = 3). The high concentration of NaCl does not poison the electrode. Its application for the detection of glucose in human blood serum sample shows good agreement with the results obtained from commercial glucose meter. The Langmuir isothermal theory was employed to fit the obtained calibration curve. The mechanisms for the glucose oxidation promoted by Co(3)O(4) nanofibers and the good selectivity against uric acid and ascorbic acid at an applied potential of +0.59 V vs. Ag/AgCl were also proposed. These results demonstrate that Co(3)O(4) nanofibers have great potential applications in the development of sensors for enzyme-free detection of glucose. Published by Elsevier B.V.