A novel method to fabricate transmembrane protein (TP) embedded lipid bilayers using microcontact printing and applying proteoliposomes to different types of substrates, has been developed. The electrostatic interaction between the negatively charged proteoliposome and the substrate, which had been positively functionalized by microcontact printing, allowed the formation of TP-embedded, patterned lipid bilayers. The positively charged amino functional group on the substrate did effectively attract the negatively charged vesicles, inducing them to be adsorbed and subsequently ruptured to form a giant mosaic lipid bilayer, resulting in an immobilized TP-embedded lipid layer precisely on the targeted patterns, which were backfilled with a zwitterionic lipid bilayer. The rapid and highly selective recognition of the charged liposomes was visualized, and the biological functions of the TPs in the lipid matrix were also observed.