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In this study, a new lactose biosensor has been developed in which the 3,4-ethylenedioxythiophene/thiophene (EDOT/Th) copolymer is used as a transducer. The EDOT/Th copolymer was deposited on the glassy carbon electrode to be used as the working electrode. In addition to the working electrode, the three-electrode system was used in both the electrochemical synthesis and in the biosensor measurements. Lactase (β-galactosidase) that catalyzes the breakdown of lactose into monosaccharides (glucose and galactose) and galactose oxidase that catalyzes the oxidation of the resulting galactose were attached to the copolymer by a cross-linker on the modified working electrode. The response of the enzyme electrode to lactose was determined by cyclic voltammetry (CV) at +0.12 V. Enzyme electrode optimization parameters (pH, temperature, enzyme concentration, etc.) were performed. Fourier transform infrared spectroscopy, scanning electron microscopy and CV methods were used to support copolymer formation. In addition, the characteristics of the enzyme electrode prepared in this study (Km, 0.02 mM; activation energy Ea, 38 kJ/mol; linear working range, up to 1.72 mM; limit of detection, 1.9 × 10−5 M and effects of interferents [uric acid and ascorbic acid]) were determined.