Biopolymer polyelectrolyte multilayers are a commonly studied soft matter system for wound healing applications due to the biocompatibility and beneficial properties of naturally occurring polyelectrolytes. In this work, a popular biopolymer, chitosan, was combined with the lesser known polysaccharide, fucoidan, to create a multilayer film capable of sequestering growth factor for later release. Fucoidan has been shown to act as a heparin-mimic due to similarities in the structure of the two molecules, however, the binding of fibroblast growth factor-2 to fucoidan has not been demonstrated in a multilayer system. This study assesses the ability of fucoidan to bind fibroblast growth factor-2 within a fucoidan/chitosan polyelectrolyte multilayer structure using attenuated total internal reflectance infrared spectroscopy and quartz crystal microbalance with dissipation monitoring. The fibroblast growth factor-2 was sequestered into the polyelectrolyte multilayer as a cationic layer in the uppermost layers of the film structure. In addition, the diffusion of fibroblast growth factor-2 into the multilayer has been assessed.