The covalent immobilization of biomolecules on surfaces is of great interest for many medical and bioanalytical applications. Currently, there is a wide range of procedures available for the modification of surfaces to achieve covalent immobilization. Self-assembled monolayers (SAMs) are easy to generate, offer a high level of flexibility and can be precisely modified on a molecular level. Therefore, the use of SAMs for various fields in research and commercial applications is increasing fast. One of the most interesting applications is the utilization of such layers for the attachment of biomolecules. The emphasis of this review is the discussion of different aspects of uniform, mixed, and functionalized monolayers regarding their use for the immobilization of biological recognition molecules such as oligonucleotides (DNA, RNA) in comparison to proteins (especially antibodies and receptor molecules) for bioanalytical applications. In doing so, we focus on the generation of self-organizing monolayers either on smooth gold surfaces by attachment of thiol compounds or on silica surfaces by silanization.