An amperometric lactose biosensor was developed by immobilizing lactase (EC 18.104.22.168) and galactose oxidase (GaO) (EC 22.214.171.124) in Langmuir-Blodgett (LB) films of poly(3-hexyl thiophene) (P3HT)/stearic acid (SA) for estimation of lactose in milk and its products to prevent "lactose intolerance". The enzyme immobilized LB film was used as working electrode and platinum as reference electrode. The enzyme electrodes show a linearity 1-6 g/dL of lactose and have a shelf life more than 120 days. The reusability of electrode was found ten times with 3% loss in current response. The enzyme electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and kinetic parameters such as pH, temperature and stability. The working electrode may be used for the estimation of lactose/galactose in food and biological fluids.