A disposable amperometric biosensor, based on a screen-printed carbon electrode (SPCE) coated with antibodies, has been prepared and evaluated for measuring progesterone in cow's milk. This immunosensor was employed in a competitive assay involving progesterone labelled with alkaline phosphatase. Initially, phenylphosphate was used as the enzyme substrate and the product of the reaction, phenol, was detected by chronoamperometry at +0.70 V vs saturated calomel (SCE). However, this approach proved unsatisfactory, since an unidentified component of cow's milk adsorbed to the electrode and underwent a variable oxidation at this potential. This resulted in a calibration curve which did not have the characteristic sigmoidal shape. An alternative enzyme substrate, 4-aminophenolphosphate, was therefore examined, the product of the reaction, 4-aminophenol, being detectable at +0.20 V vs SCE; at this lower potential, the interferent in milk did not undergo oxidation. Consistently low background currents were obtained, enabling construction of a sigmoidal-shaped calibration curve for progesterone concentration in the milk samples investigated. Progesterone levels determined by the proposed electrochemical immunoassay compared favourably with a colorimetric immunoassay method.