An amperometric biosensor was developed to estimate galactose in human blood serum. Monolayers of poly(3-hexyl thiophene) were placed on glass plates coated with indium tin oxide formed by dispensing a mixed solution of stearic acid in chloroform on to a water sub-phase. Galactose oxidase was mixed with poly(3-hexyl thiophene)/stearic acid in chloroform and dispensed on to the air-water interface of Langmuir–Blodgett trough. These monolayers were transferred on to glass plates which were used as working electrodes with platinum as a reference electrode. The amperometric galactose biosensor thus fabricated had a linear response from 0.05 to 0.5 g galactose l−1 in blood serum. The normal level in blood is < 0.05 g galactose l−1 in adults and 0–0.2 g galactose l−1 in infants. In case of galactosemia, this increases to above 0.2 g galactose l−1 in infants.