The formation of fucoidan/chitosan-based polyelectrolyte multilayers (PEMs) has been studied with in situ Fourier transform infrared (FTIR) spectroscopy. Attenuated total reflectance (ATR) FTIR spectroscopy has been used to follow the sequential build-up of the multilayer, with peaks characteristic of each polymer being seen to increase in intensity with each respective adsorption stage. In addition, spectral processing has allowed for the extraction of spectra from individual adsorbed layers, which have been used to provide unambiguous determination of the adsorbed mass of the PEM at each stage of formation. The PEM was seen to undergo a transition in growth regimes during build-up: from supra-linear to linear. In addition, the wettability of the PEM has been probed at each stage of the build-up, using the captive bubble contact angle technique. The contact angles were uniformly low, but showed variation in value depending on the nature of the outer polymer layer, and this variation correlated with the overall percentage hydration of the PEM (determined from FTIR and quartz crystal microbalance data). The nature of the hydration water within the polyelectrolyte multilayer has also been studied with FTIR spectroscopy, specifically in situ synchrotron ATR FTIR microscopy of the multilayer confined between two solid surfaces. The acquired spectra have enabled the hydrogen bonding environment of the PEM hydration water to be determined. The PEM hydration water is seen to have an environment in which it is subject to fewer hydrogen bonding interactions than in bulk electrolyte solution.