A direct, low cost method to determine the concentration of lactose is an important goal with possible impact in various types of industry. In this study, a biosensor is reported which exploits the specific interaction between lactose and the enzyme β-Galactosidase (β-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. The biosensor was made with β-Gal immobilized in layer-by-layer (LbL) films with the polyelectrolyte poly(ethylene imine) (PEI) on an indium tin oxide (ITO) electrode modified with a layer of Prussian Blue (PB). With an ITO/PB/(PEI/PVS)1(PEI/β-Gal)30 architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 μA mmol-1 cm-2 and detection limit of 1.13 mmol L-1, which is sufficient for detecting lactose in milk and for clinical exams. Detection occurred via a cascade reaction involving glucose oxidase (GOx) titrated as electrolytic solution in the electrochemical cell, while PB allowed for operation at 0.0 V vs. saturated calomel electrode, thus avoiding effects from interfering species. Sum-Frequency Generation (SFG) spectroscopy data for the interface between the LbL film and a buffer containing lactose indicated that β-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.