The introduction of electrostatic layer-by-layer (LbL) self-assembly has shown broad biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, and drug delivery systems. Multiple assembly polyelectrolytes and proteins are based on electrostatic interaction between oppositely charged layers. The film architecture is precisely designed and can be ontrolled to 1-nm precision with a range from 5 to 1000 nm. Thin films can be deposited on any surface including many widely used biomaterials. Microencapsulation of micro/nanotemplates with multilayers enabled cell surface modification, controlled drug release, hollow shell formation, and nanobioreactors. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas.