The aim of the study presented here is to develop a biosensor based on mushroom (Agaricus bisporus) tissue homogenate for sensitive and economical determination of sulfite in foods. The working principle of the biosensor is based on an inhibition effect of sulfite on polyphenol oxidases in mushroom. Mushroom tissue homogenate was immobilized by gelatin and glutaraldehyde on a Clark-type oxygen electrode. Some optimization studies related to the bioactive layer components and working conditions were identified. The biosensor was applied to the food samples. The biosensor reported here was successfully allowed to analyze sulfite, which was a food additive in real food samples.