We report herein the design of a novel biosensor sensing strategy for sensitive detection of acetylcholine based on PAMAM-Sal dendrimer. PAMAM-Sal, salicylaldehyde and PAMAM dendrimer have been synthe- sized by means of condensation. It has been determined that PAMAM-Sal dendrimer was formed by the formation of Schiffbase with FT-IR, 1 H NMR and UV spectra. In addition, the structure has been sup- ported by elemental analysis. Later, a bienzymatic biosensor system has been developed. The bienzymatic biosensor system with acetylcholine esterase (AChE) and choline oxidase (ChO) was prepared with car- bon paste electrode modified with PAMAM-Sal for determination of the amount of acetylcholine. Acetyl- choline esterase and choline oxidase enzymes were immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H 2 O 2 at + 0.4 V vs. Ag/AgCl. The linear working range for acetylcholine determi- nation of biosensor was identified. The effects of pH and temperature on the response of the biosensor were examined. Reusability and storage stability of the biosensor were determined. Interference effects of interferants which might be in biologic media on the response of the biosensor were also studied.