Layer-by-layer (LbL) assembly has attracted much interest because of its ability to provide nanoscale control over film characteristics and because of a wide choice of available materials. The methods of LbL not only determine the process properties, but also directly affect film properties. In this review, we will discuss LbL methodologies that have been used in biomedical fields. Special attention is devoted to different properties arising from methods that allow for diverse biomedical applications, ranging from surface modification to tissue engineering. We conclude with a discussion of the current challenges and future perspectives.