A lactose biosensor was developed by immobilizing lactase and galactose oxidase in a polyvinyl formal membrane and was attached to the oxygen electrode of a dissolved oxygen analyzer for estimation of lactose in milk and food products. The enzyme immobilized polyvinyl formal membrane was characterized by atomic force microscopy. The biosensor showed the linearity for 1-7 g dl(-1) of lactose and can be reused for up to 20 measurements. The effects of pH, temperature and the stability of the immobilized lactase and galactose oxidase in PVF membrane were also studied. The enzyme membrane was found stable up to 35 degrees C and had a shelf-life of more than three months at 4 degrees C.