To achieve an atomically clean surface of single-crystal aluminum nitride (AlN) substrates, this study systematically evaluated the effects of each step in ex situ wet chemical cleaning (solvent, piranha solution, HF, HCl) and in situ hydrogen annealing. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) analyses revealed that while the combination of solvent and piranha solution exposed step morphology, its effectiveness in removing organic contaminants was limited. HF cleaning efficiently removed the oxide layer but introduced fluorine residues, whereas HCl cleaning left no chlorine residues but exhibited lower efficiency in oxide removal. In situ hydrogen annealing significantly reduced carbon and oxygen contamination, albeit accompanied by a transformation of the surface morphology from step to island mode. By modulating the low V/III ratio during low-temperature metal–organic chemical vapor deposition (MOCVD) growth, a controlled transition from 3D island growth to 2D step-flow growth was achieved. This research provides a basis for optimizing AlN substrate surface treatment, offering important insights for advancing nitride-based optoelectronic and power devices.