The unique electronic and chemical structures of carbon nanotubes (CNTs) have well enabled their applications in electrochemistry and electroanalytical chemistry; however, the difficulty in reproducibly confining CNTs onto substrate electrodes, particularly onto microelectrodes, still remains to be addressed. In this study, we develop a method to reproducibly confine single-walled carbon nanotubes (SWNTs) onto carbon fiber microelectrodes (CFEs) with electrophoretic deposition (EPD) for in vivo measurement of ascorbate. Under 2.5 V, acid-treated SWNTs are uniformly deposited on CFEs. After thermal treatment at 300 °C followed by electrochemical treatment in 0.5 M H2SO4, the SWNT-sheathed CFEs exhibit good activity to accelerate the electrochemical oxidation of ascorbic acid (i.e., ascorbate, in a neutral solution) at an onset potential of -0.15 V vs Ag/AgCl and could in vivo selectively detect ascorbate. The controllable procedures employed for EPD and pretreatment avoid the deviation in the conventional manual modification methods such as drop casting and hand rolling previously used for confining SWNTs onto an electrode surface. With the electrodes prepared here, we find that level of extracellular ascorbate in the rat cortex increases by 20.4 ± 4.8% ( n = 4), relative to its basal level, within 9 min after infusion of kainic acid into the hippocampus to evoke epilepsy. This study offers a reproducible method to prepare SWNT-sheathed CFEs for in vivo monitoring ascorbate that would largely facilitate future studies on neurochemical processes of ascorbate in various physiological and pathological events.