In situ scanning tunneling microscopy (STM) has been first employed to investigate the adsorption of RB (rhodamine 13) on solid surface in solution. An I/Au(111) (iodine-modified Au(111)) surface was used as a substrate for molecular adsorption. In situ STM imaging revealed a highly ordered RB molecular adlayer. A unit cell with parameters of a = 1.71 nm and b = 2.25 nm was determined. Higher resolution STM images disclosed the structural details of the molecular adlayer. On the basis of STM results, the RB molecule is proposed to adsorb on the I/Au(111) surface by its two diethylamino functional groups in an edge-on orientation. The orientation was related to the molecular degradation.