A novel electrochemiluminescence (ECL) biosensing system was designed for the detection of acetylthiocholine chloride (ATCI) in this work. First, graphene-AuNPs-chitosan (GR-AuNPs-CS) nanocomposite, which possesses the property of intensification effect on the ECL of luminol, was electrochemically deposited on the bare GCE. Then, a biofunctional Fe3O4-TiO2-AChE-ChO biocomposite was unprecedentedly prepared which exhibited the mimic peroxidase activity of Fe3O4 and the enhancement effect of TiO2 on the ECL intensity of luminol. Subsequently, the substrate ATCI was hydrolyzed by AChE of AChE-ChO multiple enzymes to generate thiocholine, which was then catalyzed by ChO to produce H2O2 in situ. H2O2, as the coreactant of luminol-ECL system can enhance the ECL intensity of luminol. On the basis of this principle and smart integration of the above nanocomposites under the optimal conditions, the resulting ECL biosensor for ATCI showed wide linear range from 6.7 nM to 0.92 mM with detection limit (signal-to-noise ratio of 3) of 2.2 nM. The excellent performance of the biosensor makes this biosensing system suitable and promising in the practical application of choline type of biomass. (C) 2014 Elsevier Ltd. All rights reserved.