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Self-assembly methods are being studied for construction of nanoscale chemical sensors employing coimmobilized enzymes and indicator dyes. This paper describes the assembly of the catalytic enzyme lactate oxidase in multilayer films on colloidal templates via layer-by-layer self-assembly, which is a step toward achieving nanoengineered biosensors. The architecture of the resulting films was characterized using quartz crystal microbalance and zeta potential analysis, and catalytic activity was characterized colorimetrically. The tailored activity of the functional nanofilms was proportional to the number of enzyme layers deposited during assembly, which provides a basis for designing sensors with specific interactions.