Supported lipid membranes are widely used platforms which serve as simplified models of cell membranes. Among numerous methods used for preparation of planar lipid films, self-assembly of bicelles appears to be promising strategy. Therefore, in this paper we have examined the mechanism of formation and the electrochemical properties of lipid films deposited onto thioglucose-modified gold electrodes from bicellar mixtures. It was found that adsorption of the bicelles occurs by replacement of interfacial water and it leads to formation of a double bilayer structure on the electrode surface. The resulting lipid assembly contains numerous defects and pinholes which affect the permeability of the membrane for ions and water. Significant improvement in morphology and electrochemical characteristics is achieved upon freeze–thaw treatment of the deposited membrane. The lipid assembly is rearranged to single bilayer configuration with locally occurring patches of the second bilayer, and the number of pinholes is substantially decreased. Electrochemical characterization of the lipid membrane after freeze–thaw treatment demonstrated that its permeability for ions and water is significantly reduced, which was manifested by the relatively high value of the membrane resistance.