Atomic force microscopy (AFM) of biomolecular processes at the single-molecule level can provide unique information for understanding molecular function. In AFM studies of biomolecular processes in solution, mica surfaces are predominantly used as substrates. However, owing to its high surface charge, mica may induce high local ionic strength in the vicinity of its surface, which may shift the equilibrium of studied biomolecular processes such as biopolymer adsorption or protein DNA interaction. In the search for alternative substrates, we have investigated the behavior of adsorbed biomolecules, such as plasmid DNA and E. coli RNA polymerase 030 subunit holoenzyme (RNAP), on highly oriented pyrolytic graphite (HOPG) surfaces modified with stearylamine and oligoglycine-hydrocarbon derivative (GM) monolayers using AFM in solution. We have demonstrated ionic strength -dependent DNA mobility on GM HOPG and nativelike dimensions of RNAP molecules adsorbed on modified HOPG surfaces. We propose an approach to the real-time AFM investigation of transcription on stearylamine monolayers on graphite. We conclude that modified graphite allows us to study biomolecules and biomolecular processes on its surface at controlled ionic strength and may be used as a complement-to mica in AFM investigations.