A rapid and simple approach of lactose analysis is proposed based on 3rd generation amperometric biosensors employing cellobiose dehydrogenase (CDH) from Trametes villosa or Phanerochaete sordida immobilised on screen-printed carbon electrodes (SPCEs). After optimisation of the working conditions of the biosensors - pH of the carrier buffer, flow rate and applied potential - the sensors were able to detect lactose in a concentration range between 0.5-200 mu M and 0.5-100 mu M employing T. villosa and P. sordida CDH, respectively. The limit of detection is 250 nM (90 mu g/L) for both. Biosensors based on SPCEs modified with multiwalled carbon nanotubes showed a higher sensitivity than unmodified SPCEs. Crosslinking with glutaraldehyde or poly(ethyleneglycol)diglycidyl ether improved not only the stability but also the analytical response. The developed sensor has been successfully applied for the determination of lactose in dairy (milk with different percentages of fat, lactose-free milk and yogurt) with a good reproducibility (RSD = 1.5-2.2%). No sample preparation except a simple dilution process is needed. The biosensor is easy to make and operate, is inexpensive and reveals a high sensitivity and reliability. (C) 2009 Elsevier Ltd. All rights reserved.