The success of single-molecule (SM) experiments critically depends on the functional immobilization of the biomolecule(s) to be studied. With the continuing trend of combining SM fluorescence with SM force experiments, methods are required that are suitable for both types of measurements. We describe a general protocol for the site-specific and covalent coupling of any type of biomolecule that can be prepared with a free thiol group. The protocol uses a poly(ethylene glycol) (PEG) spacer, which carries an N-hydroxy succinimide (NHS) group on one end and a maleimide group on the other. After reacting the NHS group with an amino-functionalized surface, the relatively stable but highly reactive maleimide group allows the coupling of the biomolecule. This protocol provides surfaces with low fluorescence background, low nonspecific binding and a large number of reactive sites. Surfaces containing immobilized biomolecules can be obtained within 6 h.