The covalent attachment of Fab′ fragments of polyclonal anti-human IgG to a polymerizable lipid with a terminal linker group was examined by means of quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and atomic force microscopy (AFM). The linker lipid was embedded in a monolayer of dilinoeloylphosphatidylethanolamine. Both monomeric and cross-linked biofunctionalized monolayers were studied. Atomic force microscope images showed that the monomeric monolayer consisted of large holes when it was deposited on a solid substrate, while the cross-linked monolayer appeared as a planar two-dimensional film. The ability of the biofunctionalized monolayer to bind proteins decreased with UV-irradiation time. However, an increase in the linker lipid concentration in the lipid matrix increased the protein-binding efficiency. A comparison between QCM and SPR measurements indicated that the QCM measurements overestimated the binding efficiency of immobilized Fab′ fragments toward hIgG. AFM images visualized the topographical changes of the different stages of the monolayer incubation in Fab′, BSA, and hIgG protein solutions.