We present a novel concept for the creation of lipid vesicle microarrays based on a patterning approach termed Molecular Assembly Patterning by Lift-off (MAPL). A homogeneous MAPL-based single-stranded DNA microarray was converted into a vesicle array by the use of vesicles tagged with complementary DNAs, permitting sequence-specific coupling of vesicles to predefined surface regions through complementary DNA hybridization. In the multistep process utilized to fulfill this achievement, active spots consisting of PLL-g-PEGbiotin with a resistant PLL-g-PEG background, as provided by the MAPL process, was converted into a DNA array by addition of complexes of biotin-terminated DNA and NeutrAvidin. This was then followed by addition of POPC vesicles tagged with complementary cholesterol-terminated DNA, thus providing specific coupling of vesicles to the surface through complementary DNA hybridization. Quartz crystal microbalance with dissipation (QCM-D) and optical waveguide lightmode spectroscopy monitoring were used to optimize the multistep surface modification process. It was found that the amount of adsorbed biotinDNA-NeutrAvidin complexes decreases with increasing molar ratio of biotinDNA to NeutrAvidin and decreasing ionic strength of the buffer solution. Modeling of the QCM-D data showed that the shape of the immobilized vesicles depends on the amount of available anchoring groups between the vesicles and the surface. Fluorescent microscopy images confirmed the possibility to create well-defined patterns of DNA-tagged, fluorescently labeled vesicles in the micrometer range.