Alternate layer-by-layer (LbL) adsorption has received much attention as an emerging methodology. Biocompatibility is the most prominent advantage of the LbL assembly process because the technique employs mild conditions for film construction. Most enzymes, especially water-soluble ones, have charged sites at their surfaces so that electrostatic LbL adsorption is suitable for construction of various protein organizations. In this review chapter, we summarize recent developments on enzyme-encapsulated LbL devices and their related functions where “encapsulated” does not always entail entrapment within spherical structures but generally includes immobilization of enzymes within the LbL structures. Recent examples, with various functions such as reactor sensors and medical applications, are described within a classification of structural types, i.e., thin films and spherical capsules. In addition to conventional applications, advanced systems including integration of LbL structures into advanced devices such as microchannels, field effect transistors, and flow injection amperometric sensors are introduced as well as recent developments in hybridization of LbL assemblies with functional nanomaterials such as carbon nanotube, dendrimers, nanoparticles, lipid assemblies, and mesoporous materials, all of which can enhance bio-related functions of LbL assemblies.