An amperometric biosensor prepared by immobilization of P-galactosidase and glucose oxidase (GOD) onto a glassy carbon electrode coated with mercury thin film for lactose has been developed. The enzymes were immobilized by gelatin and glutaraldehyde onto mercury thin film electrode easily. The chronoamperometric detection of lactose was carried out at -0.2 V (versus Ag/AgCl) in 0.05 M citrate buffer (pH 5.5). Optimum pH and temperature, and thermal stability were investigated. Interference effects of some sugars and repeatability of the biosensor were also studied. The response to lactose was found to be linear range between 1 x 10(-4) and 3.5 x 10(-3) M. The biosensor was applied to the determination of lactose in milk samples and the results obtained with the biosensor were validated by Somogyi-Nelson method. (c) 2005 Elsevier B.V. All rights reserved.