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AIM: To determine the concentration of acetylcholine (ACh) in amyloid beta-protein (Aβ) treated rats and offer a method determining ACh as well. METHODS: A 1-month combination of extrats of ginseng and ginkgo biloba(Naoweikang) ig administration to rats was performed daily after bilateral injection of Aβ1-40 (4 g/L, 1 μL for each side) into hippocampus. After decollation, homogenizing, and centrifuging and extracting, a high pressure liquid chromatographic (HPLC) method using electrochemical detection (ECD) combined with two immobilized enzyme reactors was used to determine ACh in rat whole brain. RESULTS: With a mobile phase consisting of disodium hydrogen orthophosphate, tetramethylammonium chloride (TMAC1), octanesulfonic acid sodium salt (OSA) and"Reagent MB" at a final pH of 8.0, ACh was determined while removing the interfering choline in less than 10 min at a flow rate of 0.35 mL/min on a platinum (Pt) working electrode at a potential of +300 mV vs a solid-state palladium (Pd) reference electrode. Linear regression analysis of peak area vs concentration demonstrated linearity in the 28.01 to 1400.06 μg/L injection range. The r-value was 0.9978. The limit of detection (LOD) is 0.28 ng on column. ACh in whole brain decreased by 20.34 % (from 162.1±32.7 to 134.7±14.0 μg/L, P＜0.05) after bilateral injection of Aβ into rat hippocampus. After Naoweikang administration (31 and 15.5 mg/kg, respectively), ACh increased by 19.97 % (from 134.7+14.0 to 161.6+26.2 μg/L, P＜0.05) and 18.56 % (from 134.7+14.0 to 159.7+22.9 μg/L, P＜0.05), respectively. CONCLUSION: Naoweikang significantly increased the level of ACh in whole brain of Aβ treated rats. And a sensitive, selective and reliable method for routinely determining ACh in rat whole brain was established in this study.