Anew biosensor based on mushroom (Agaricus bisporus) tissue homogenate was developed for voltammetric determination of ethanol. Mushroom (Agaricus bisporus) was homogenated and immobilized onto the glassy carbon electrode via gelatine and cross-linked by glutaraldehyde to construct an electrochemical tissue homogenate based biosensor system. The homogenate contains alcohol oxidase (E.C 1.1.3.13) enzyme and it catalysis the degredation of ethanol to acetaldehyde and hydrogen peroxide. Assay principle of the biosensor based on the detection of reduction peaks of oxygen by using cyclic voltammetry at potentials between 0.0 and +0.7 V. To obtain a standard graphe for ethanol differential pulse (DP) method was used at potentials between +0.1 and +0.4 V. The electrode response depends linearly on ethahol concentration between 1.0 and 10.0 mM. Phosphate buffer (pH 7.0, 50 mM) and 30°C were obtained as the optimum conditions. In characterization studies of the biosensor some parameters such as; repeatability, substrate specificity, interference effects of some substances on the biosensor response were investigated. Finally, the proposed system was applied to ethanol detection in different wine and beer samples.