A new lactose biosensor was developed by preparing a suitable copolymer of polypyrrole and poly(3,4-ethylenedioxythiophene) synthesized using the electropolymerization method. Pyrrole and 3,4-ethylenedioxythiophene monomers were deposited in the presence of sodium dodecylbenzene sulphonic acid on a platinum disc electrode, which was used as the working electrode. The sensor is based on the serial reactions of -galactosidase and galactose oxidase immobilized on a copolymer-modified platinum disc electrode. Successful synthesis of the enzyme-immobilized copolymer was confirmed by FT-IR spectrometry, SEM, and electrochemical analysis. The response of the enzyme electrode to lactose was determined by cyclic voltammetry at+0.40 V. The response time of the biosensor was found to be from 8 to 10 s, and the upper limit of the linear working portion was found to be at a lactose concentration of 2.30 mM with a detection limit of 1.4 x 10(-5) M. The apparent Michaelis-Menten constant was found to be 0.65 mM of lactose. The effects of interferents were also investigated. Lactose concentrations determined by the biosensor were in good agreement with those measured by the reference methods. Our results show that the developed biosensor has a significant potential to the determination of lactose concentration in milk. POLYM. ENG. SCI., 58:839-848, 2018. (c) 2017 Society of Plastics Engineers