Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, and biophysics, and for controling biological processes. The layer‐by‐layer (LbL) assembly technology is a highly versatile methodology introduced 30 years ago, which consists in assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self‐assembled films. In view of its versatility, simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and of supporting carriers, and to modify the film mechanical properties via crosslinking. In this review, we focus on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. We highlight possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering. In addition, we present the most recent technological developments in the field of films construction, such as high content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL.This article is protected by copyright. All rights reserved