Multienzyme microstructures of glucose oxidase (GOx) and horseradish peroxidase (HRP) were prepared by layer-by-layer deposition inside microfluidic networks on glass substrates in order to allow both site-specific deposition and control of the amount of immobilized enzymes. The obtained microstructures were characterized by scanning force microscopy for the topography of the deposited layers. The local enzyme activity was characterized by the substrate-generation/tip-collection mode and the enzyme-mediated feedback mode of the scanning electrochemical microscope (SECM). These measurements provided quantitative information about the immobilized enzyme activity as a basis for adjusting enzyme loading for multienzyme structures that realize logical operations based on enzymatic conversions. Information about local HRP activity can also be obtained by optical readout using an Amplex UltraRed fluorgenic substrate and reading with a confocal laser scanning microscope with a much higher repetition rate for image acquisition. Using these principles, a layout with HRP and GOx microstructures was realized that showed the functionality of an OR Boolean logic switch.