To detect a low level of acetylcholine (Ach) output in the dialysate of rat frontal cortex, osmium-peroxidase redox polymer modified gold-ring disk electrode (Os-HRP-RDE) was fabricated in high-performance liquid chromatography (HPLC)-electrochemical detection (ECD). In comparison with platinum electrode, the sensitivity of acetylcholine detection by Os-HRP-RDE was stable during a day, the coefficient of variation was 0.95%, and the decrease of the sensitivity for 1 week was 5.4%. The Os-HRP-RDE could detect lower than 3fmol of acetylcholine. The present technique, in the absence of acetylcholinesterase (AchE) inhibitor, is useful to facilitate physiological investigations of cholinergic neuronal activity in the brain.