The design of functional polymer multilayer thin films with nanometer scale control is of great interest for biomedical applications such as tissue engineering, targeted drug delivery, controlled release system, and regenerative medicine. Various functions and properties of polymer thin films can be easily programmed and realized by the layer-by-layer assembly strategy, which is a facile and versatile deposition method to prepare well-defined biomedical multilayer platforms due to its benign process to prepare films under mild conditions and the capability of incorporating bioactive materials at a desired location within the films. Particularly, the fine tuning of physicochemical and biological properties of multilayer thin films is significantly important for designing novel biomedical platforms capable of adjusting the cellular functions. In this review, we focus on the overall background of the layer-by-layer assembly as well as the tuning of multilayer film properties and the programming of biological functions into the polymer thin films with a view on the control of cellular functions. Furthermore, we highlighted the recent achievements toward the design of novel biomedical platforms based on functionalized polymer multilayer thin films.