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An enzyme electrode for D-galactose determination was prepared by fixation of a carrier with immobilized galactose oxidase (E.C. 1.1.3.9) or coimmobilized galactose oxidase and catalase (E.C. 1.11.1.6) to a Clark-type oxygen sensor. The enzymes were immobilized either on a partially hydrolyzed nylon mesh or on a native collagen membrane using the Ugi reaction with cyclohexyl isocyanide and glutaraldehyde. The biosensors were characterized by the specific activity of the immobilized galactose oxidase, the apparent Michaelis constant KM(app.), and the stability expressed by time and a number of the performed analyses. The substrate specificity of the biosensor and the effect of pH and temperature of the reaction mixture on the response magnitude were also tested. The prepared biosensor was used for the determination of D-galactose content in samples of blood plasma and serum of patients with suspected galactosemia.