A glucose oxidase-based biosensor was developed for the determination of alpha-amylase activity. The determination method is based on monitoring the decrease in dissolved oxygen concentration related to the starch concentration, for which starch gives a reaction with alpha-amylase. Optimization parameters, including glucose oxidase amount, gelatin amount, and glutaraldehyde percentage for cross-linking, were investigated. The effects of pH, buffer system, and temperature on the biosensor system were also investigated. The biosensor had a linear relation to alpha-amylase activity and good measurement correlation between 0.66 and 9.83 U/ml. In sample analysis studies, alpha-amylase activity in baker's yeast was determined by the biosensor.