A biosensor based on a glass electrode was developed to determine free sulfur dioxide in wine. The biosensor consists of a microbial membrane of Thiobacillus thiooxidans JCM7814 and a flat glass electrode. A porous gas-permeable membrane was incorporated in the biosensor system to separate free sulfur dioxide from bound sulfur dioxide and to avoid the buffer action of the sample solution. This biosensor gave a linear relationship between the pH decrease and concentration of sulfur dioxide up to 50 mg/liter with a response time of 20 min. The limit of detection for sulfur dioxide was 5.0 mg/liter, and the life-time of the microbial membrane was ca.30 days at 4°C. The biosensor could specifically determine free sulfur dioxide in wine, and was not influenced by temperature or oxygen concentration. The relative standard deviations were 7.41 % for red wine and 5.00% for white wine.