Selective and reliable method is of great importance for in vivo analysis in the complicated brain. In this work, we developed a new approach for detection of ascorbic acid (AA) based on a new nitrogen-doped nanotube fiber (NCNF) microelectrode. After electrochemical pre-treatment, the NCNF microelectrode (e-NCNF) demonstrated high electrocatalytic capability for AA oxidation with a low working potential, 0 V, resulting in high selectivity for detection of AA level against other biological species in brain, as well as producing high-sensitivity. Moreover, the e-NCNF was directly assembled by carbon nanotube bundles, free from-the post-modification, thus showing high reproducibility, in which the deviation of anodic current intensity of six electrodes prepared with same method did not exceed 4% (RSD, n = 6). Based on the remarkable analytical performance, the e-NCNF with good biocompatibility was successfully applied to determine the AA level as 134 +/- 7 mM, in rat brain microdialysates. (C) 2016 Elsevier B.V. All rights reserved.