In the past two decades, the design and manufacture of nanostructured materials has been of tremendous interest to the scientific community for their application in the biomedical field. Among the available techniques, layer-by-layer (LBL) assembly has attracted considerable attention as a convenient method to fabricate functional coatings. Nowadays, more than 1000 scientific papers are published every year, tens of patents have been deposited and some commercial products based on LBL technology have become commercially available. LBL presents several advantages, such as (1): a precise control of the coating properties; (2) environmentally friendly, mild conditions and low-cost manufacturing; (3) versatility for coating all available surfaces; (4) obtainment of homogeneous film with controlled thickness; and (5) incorporation and controlled release of biomolecules/drugs. This paper critically reviews the scientific challenge of the last 10 years--functionalizing biomaterials by LBL to obtain appropriate properties for biomedical applications, in particular in tissue engineering (TE). The analysis of the state-of-the-art highlights the current techniques and the innovative materials for scaffold and medical device preparation that are opening the way for the preparation of LBL-functionalized substrates capable of modifying their surface properties for modulating cell interaction to improve substitution, repair or enhancement of tissue function.