This work constitutes the first biocentri-voltammetric application for acetylcholinesterase (AChE) activity assay. After the optimization of working conditions like reaction time, centrifugation speed and time and enzyme amount, the analytical characteristics were investigated. As a result, the linear range of 1.0-350 mu M was obtained with an equation of y = 0.0004x + 0.0163. The correlation coefficient is equal to (R-2) 0.99 +/- 0.001815 (n = 3) and the RSD value was calculated as 3.6% (n = 4). Then Donepezil-based drug analysis was also conducted by using the developed method and an inhibition of 80% was calculated.