The goal of this work was to design a system incorporating gramicidin (gA) into a polysulfone (Psf) membrane. The strategy involved immobilization of gA on the surface of two types of magnetite nanoparticles (MNPs) dispersed in a polymeric matrix. Membranes have been treated with surfactant Triton X100 (TRX) and surfactant/protein micelles (TRX/gA). MNP morphology was studied using transmission electron microscopy, and the chemical structure was confirmed by Fourier transform infrared analysis. Membrane morphology was observed under environmental scanning electron microscopy; water interactions were tested using static contact angle and water uptake, whereas ion transport was evaluated by means of a permeability test and current-voltage experiments. Depending on the type of nanoparticles used, immobilization led to either higher or lower membrane hydrophilicity but did not influence their morphology. Protein immobilization resulted in enhanced ion diffusion properties and membrane selectivity. This novel method of fabricating materials has a tremendous potential for use in ion-exchange applications, such as fuel cells or nanofiltration.