This paper reports the studies in 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/AgCI film as the reference electrode. The silver bands acted as the conductor under the electrodes for the purpose of minimizing the interior resistance of electorde system. The resulting biosensor was constructed by immobilizing the extracted protein of myshroom 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