The properties of a glucose biosensor made by immobilization of glucose oxidase onto gelatin in a layer of electrochemically deposited polyaniline have been investigated. Glucose oxidase was immobilized within gelatin cross-links with chromium(III) acetate. The glucose oxidase biosensor was developed by forming a polyaniline-deposited electrode surface as support for the immobilized enzyme gel, in order to increase its durability. The polyaniline/gelatin/glucose oxidase biosensor has been characterized using chemical and electrochemical methods. Temperature, pH, cross-linking agent concentration, enzyme concentration, kinetic properties, reusability and the effect of electro-active compounds were among the parameters studied. The response time of the glucose oxidase biosensor is 90 s, the detection limit is below 1 mmol/dm3 and the sensor can be used 20 times within a 2-month period without losing its stability.