Ascorbic acid (AA) levels are closely correlated with physiological and pathological events in brain diseases, but the mechanism remains unclear, mainly due to the difficulty of accurately analyzing AA levels in live brain. In this study, by engineering tunable defects and oxygen-containing species in carbon nanotubes, a novel aligned carbon nanotube fiber was developed as an accurate microsensor for the ratiometric detection of AA levels in live rat brains with Alzheimer's disease (AD). AA oxidation is greatly facilitated on the fiber surface at a low potential, leading to high sensitivity as well as high selectivity against potential sources of interference in the brain. Additionally, an unexpected, separate peak from the fiber surface remains constant as the AA concentration increases, enabling real-time and ratiometric detection with high accuracy. The results demonstrated that the AA levels were estimated to be 259 ± 6 μM in cortex, 264 ± 20 μM in striatum, and 261 ± 21 μM in hippocampus, respectively, in normal condition. However, the overall AA level was decreased to 210 ± 30 μM in cortex, 182 ± 5 μM in striatum, and 136 ± 20 μM in hippocampus in the rat brain model of AD. To the best of our knowledge, this work is the first to accurately detect AA concentrations in the brains of live animal model of AD.