In situ scanning tunneling microscopy was employed to study the pretreatment method of Fe(110) surface and the adlayer structure of o-aminothiophenol (OATP) formed on it in 0.1 M NaClO4. A preparation method consisting of chemical etching and electrochemical annealing steps was primarily determined, which can consistently give atomically flat terraces on Fe(1 10) surface. This way of preparation could open up new perspectives for the investigations of surface structural properties on Fe that were normally not accessible because of easy oxidation and contamination outside ultrahigh vacuum. For the first time, the atomic resolution Fe(110) surface and the molecular resolution adlayer on it were imaged in solution. OATP molecules adsorbed on the Fe(110) surface and formed well-ordered p(2 x 2) structures with a coverage of 0.25.