An amperometric biosensor was designed using immobilizing alcohol oxidase in conducting polymer of poly 4-(2,5-di(thiophen-2-yl)-1H-pyrrole-1-1) benzenamine matrix via crosslinking with glutaraldehyde on platinum electrode. An electrochemical technique was used for the polymerization of conducting polymer onto the electrode. Carbon nanotubes and gold nanoparticles were also added to the biosensing system to observe the effects of nanoparticles. The proposed biosensor was characterized and optimized in terms of conducting polymer thickness, enzyme loading, pH, nanoparticle effects, linear range,, repeatability and operational stability. The proposed biosensor showed good linear range, and repeatability as well as high operational stability. The proposed biosensor optimized with the conditions of potassium phosphate buffer (50 mM, pH 7), 20 cycles of SNSNH2, 1.5 mu L AOx (1.56U), 1% glutaraldehyde and -0.7 V applied potential. Linear analytical range was obtained between 0.1-5 mM ethanol and after 6 hours with 25 measurements pSNSNH(2)-AOx biosensor lost only 7% of its activity. The biosensing system was successfully applied to real samples vodka and whisky with good recovery.