A layer-by-layer structure of enzyme multilayers composed of glucose oxidase (GOx) or lactate oxidase (LOx) and ascorbate oxidase (AOx) was prepared on the surface of a platinum electrode. The amperometric response to glucose or lactate was studied in the presence of ascorbic acid as a possible interference. An alternating and repeated deposition of avidin and the biotin-labeled enzymes resulted in the layer-by-layer structure of GOx/ AOx and LOx/AOx multilayers. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of monomolecular layers of the proteins. The GOx/AOx and LOx/AOx enzyme multilayers were useful to eliminate ascorbic acid interference in the glucose and lactate biosensors, because ascorbic acid can be converted to an electrochemically inert form, dehydroascorbic acid, before being oxidized directly on the Pt electrode. Thus, the GOx/AOx or LOx/AOx multilayer-modified biosensors can be used to determine the normal blood level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). The effects of the number of the AOx layers and geometry of the enzyme layers in the multilayer on the performance characteristics of the biosensors are discussed.