In this paper, we give a short account on recent studies of layer-by-layer self-assembly of supramolecular and biomolecular films. Such films are built up from layers of macro-ions with opposing charge. A simple film can be obtained by alternating the adsorption of two components: a flexible, synthetic polycation chains and a supramolecular or biomolecular moiety. We focus on three examples, in which the second component consists either of a supramolecular metal–organic complex (MOC), a nucleic acid, or a biological membrane patch (purple membrane). While the flexible polycation chains (as well as eventual annealing layers) ensure a uniform build-up of the chain, the second macromolecular component may be used to functionalize the films. The combination of layer-by-layer self-assembly and biotechnologically relevant macromolecules may lead to new devices or biomaterial applications. To this end, precise studies of the deposition process and the film structure are needed. Here, we focus on interface sensitive scattering techniques for the structural analysis.