Nanostructured thin films of cellulose nanowhiskers derived from cotton were formed using a simple drop-coating procedure. The hydrogen-bonded cellulose films were stable in aqueous solutions and their permselective properties were probed using voltammetric techniques. The nanowhisker extraction procedure produces cellulose nanowhiskers with negatively-charged sulfate surface groups that inhibit the transfer of negatively-charged species through the nanowhisker membrane, while the diffusion of neutral species is only slightly hindered. Using rotating-disk electrode measurements, the diffusion of various species within the film was studied and it was shown that the positively-charged species, Ru(NH₃)₆³⁺, was adsorbed by the film, whereas the negatively-charged species, IrCl₆³⁻, was excluded by the film. The thermodynamics of adsorption of the positively-charged species by the cellulose nanoparticles were then studied using isotherm data. These observations open up new possibilities in electrochemical sensor development using renewable cellulosic materials as building blocks. Furthermore, charge-based permselective membranes can also be formed using free standing cellulose nanowhisker films, which offer the promise of renewable, selective membranes for separation technologies.