In this paper, a novel amperometric bienzymatic biosensor based on iron (II, III) oxide nanoparticles (Fe(3)O(4)NPs), multi-walled carbon nanotubes (MWCNTs) and chitosan (CS) modified glassy carbon electrode (GCE) was fabricated for the determination of acetylcholine (ACh). The CS was used to immobilize acetylcholine esterase (AChE) and choline oxidase (ChOx). The nanocomposites of Fe(3)O(4)NPs and MWCNTs were characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical measurements were based on the detection of enzymatically produced hydrogen peroxide (H2O2). The experimental parameters such as working potential, enzyme unit, pH and temperature were optimized. The linear ranges of the biosensor were 0.02 mu mol L-1-0.111 mu mol L-1 and 0.111 mu mol L-1-1.87 mu mol L-1. The detection limit was calculated as 0.61 nmol L-1. In addition, the amperometric biosensor showed high sensitivity, good selectivity, repeatability, reproducibility and long-term stability. The fabricated biosensor was used to determine ACh levels in serum samples. (C) 2016 Elsevier B.V. All rights reserved.