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In this work, S-layer proteins were used as an immobilization matrix to link aptamers on different solid supports. In nature, S layers operate amongst others as an immobilization matrix for exoenzymes. Consequently, they provide a biocompatible environment with different kinds of chemical groups perfect for the sequential coupling of any kind of biofunctional molecules. In addition, their nanostructure ensures a regular arrangement of these biomolecules. In biosensors, different biological recognition molecules are used. In this study, aptamers were chosen as bio-receptors. Aptamers are oligonucleotide ligands that are especially selected for high-affinity binding to target molecules. Because of their small size and stability, they exhibit a high potential as biological sensing molecules. By coupling aptamers to different surfaces or combining them with other biofunctional molecules, target binding can be detected for example optically or gravimetrically. In this work, a thrombin-binding aptamer and an ofloxacin-binding aptamer were immobilized by different chemical crosslinkers to surfaces modified with S-layer proteins. To verify the functionality of immobilized aptamers, the aptamer-target-binding was proven by Laser Induced Fluorescence Spectroscopy (LIFS), a Resonant Mirror Sensor (IAsys) and a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D), respectively. Due to their properties of building up a physiological environment on their surface, their high content of modifiable functional groups on their surface and their ability to crystallize in a nanometer thick monolayer on surfaces, S-layer proteins are suitable as biotemplates for various recognition biomolecules like enzymes, antibodies and aptamers. Hence, this paper presents with S-layer proteins an interesting alternative to existing immobilization matrices for recognition biomolecules.