A novel amperometric ethanol biosensor with alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD(+)) on polypyrrole-polyvinylsulfonate (PPy-PVS) film was accomplished. Meldola's blue (MB) was used as a mediator; it can facilitate the electron transfer that involves a chemical interaction between the electrode surface and NADH. Determination of ethanol was carried out by the oxidation of MB at -0.072 V vs. Ag/AgCl. The effects of pH and temperature were investigated and optimum parameters were found to be 7.0 and 40 degrees C, respectively. There are 2 linear parts at 1.0 mu M-10.0 mu M (R-2 = 0.993) and 0.01 mM-0.1 mM (R-2 = 0.996). The storage stability and operational stability of the enzyme electrode were also studied. The results showed that 95.7% of the response current was retained after 17 activity assays. The ethanol biosensor gave perfect reproducible finding with 4.6% relative standard deviation. The prepared biosensor retained 55.8% of initial activity after 24 days when stored in 0.1 M phosphate buffer solution at 4 degrees C. Effects of the several possible interfering substances such as ascorbic acid, citric acid, and methanol on the ethanol biosensor were investigated and the developed biosensor was tested in alcoholic beverages.