In this paper, a novel amperometric acetylcholine (ACh) biosensor with immobilization of acetylcholinesterase-choline oxidase (AChE-ChO) on polypyrrole-polyvinylsulphonate (PPy-PVS) film has been successfully developed. AChE and ChO enzymes were immobilized on PPy-PVS film by cross-linking with glutaraldehyde (GA) and bovine serum albumine (BSA). Determination of ACh was carried out by oxidation of enzymatically produced H2O2 at 0.3 V vs Ag/AgCl. The effects of pH and temperature were investigated and optimum parameters were found to be 9.0 and 65 degrees C, respectively. There were two linear parts in the region between 1.0 x 10(-8)-1.0 x 10(-7) M (R-2 = 0.994) and 1.0 x 10(-7)-1.0 x 10(-6) M (R-2=0.994). The detection limit of the biosensor was 5.0 x 10(-9) M and response time of the biosensor was 200 s. Storage stability and operation stability of the enzyme electrode were also studied. The results showed that 83.1% of the response current was retained after 18 activitiy assays. ACh biosensor gave perfect reproducible findings with 4.4% relative standard deviation. The prepared ACh biosensor retained 31% of its initial activity after 35 days storage in 0.1 M glycine buffer solution at 4 degrees C. (C) 2014 Elsevier B.V. All rights reserved.