All-trans retinoic acid (RA) has been proved to play important roles in regulating cell growth in various types of cells. Yet most experiments were performed by adding RA in solution previously. In this Article, we focus on the incorporation of RA, as a negatively charged moiety, into layered polyelectrolyte films on surfaces by means of layer-by-layer (LbL) deposition, followed by adding of capping layers to regulate the release of RA from the films. The incorporated RA was designed to release over 5 days in buffer solution. The assembly and release of RA were verified by UV and QCM results. The controlled release of RA from multilayer films can serve as a model system to study the influence of small molecules on cell growth.