Abstract This paper reports the studies on the fabrication and optimization of the disposable amperometric biosensor. An integrated three-electrode system was constructed on the nylon sheet with the imprinted thick-layer graphite film to act as the working electrode and auxiliary electrode, and the Ag/AgCl film as the reference electrode. The silver bands acted as the conductor under the electrodes for the purpose of minimizing the interior resistance of electrode system. The resulting biosensor was constructed by immobilizing the extracted protein of mushroom that contained rich polyphenol oxidase in silk fibroin membrane on a ferrocene modified working electrode. The voltammetric current of this biosensor responds to concentrations of dopamine over six orders of magnitude, with a detecting limit of 1.0×10 −8 M and a response time on the order of seconds. The relative standard deviation (R.S.D.) of this biosensor equals to 2.7%. This biosensor can keep its activity for at least 500 times use and even for a more long time under suitable condition.