This chapter provides an overview of different assembly methodologies used for the construction of multilayer architectures with biomolecules for application in sensors. Besides the use of bioaffinity interactions and covalent strategies, special attention will be paid to the electrostatic layer-by-layer technique. Different building blocks can be used for the formation of multilayers with a clear preference for polymers and nanoparticles. Among the biomolecules, enzymes and redox proteins are in focus. Because of the high importance of multilayers formed on electrodes, the chapter will concentrate on sensor systems with electrochemical transduction. Particularly advantageous are schemes that can avoid diffusible shuttling molecules between the biomolecule and the electrode and that represent artificial signal chains by exploiting direct protein-protein communication in the immobilized state.