Layer-by-layer (LbL) assembly is an easier, inexpensive, and highly versatile bottom-up methodology to modify surfaces and fabricate functional multilayer thin films and nanocomposites with fine-tuned compositions, structures, properties, and functions at the nanoscale. Since the early stages of its development, LbL technology has gathered increasing attention across different fields of application, including in the biomedical field owing to its mild processing conditions. In this chapter, we review the multitude of templates, spanning from the zero-dimensional to the three-dimensional, for shaping a diverse set of multifunctional soft-based LbL structures aiming for biomedical applications. Several examples are given on multilayered structures, including nano-to-macro particles and hollow capsules or tubes, multilayered thin films and free-standing membranes, multi-compartmentalized systems, porous scaffolds, and even dynamic living cell platforms, which can act as unprecedented building blocks to create highly complex LbL devices. We envisage that such a multitude of functional LbL devices will stimulate scientists to pursue the further development of LbL technology and foster its effective translation to practical biomedical applications.