We report here surface potential and differential electrostatic force images of DNAs, proteins, and gold nanoparticles on insulating substrates. The frequency-shift mode was essential for these experiments to satisfy enough sensitivity to use indirect modulation and to avoid unexpected charge injection into insulating substrate surface caused by tip-sample contact. It is obvious that the surface potential of insulating substrate is indefinable and influenced by treatment process and conditions. However, the potential difference between adsorbates and substrate surface is meaningful, reflecting charge or dipoles of adsorbates. We demonstrated that electrostatic force microscopy provides characteristic contrast inversion between DNA and transcription complex images reflecting the difference of electric polarizability of these molecules. These findings indicate that the electrostatic properties of individual biological molecules can be imaged on an insulator substrate during the retaining complex formation.