We report a study of protein adsorption on controlled surfaces. Hydrophilic (carboxylic acid-terminated) and hydrophobic (methyl-terminated) self-assembled monolayers (SAMs) on Au(111) surfaces were prepared at different solution temperatures and characterized by low-current scanning tunneling microscopy (STM). Protein adsorption on these SAMs was investigated by surface plasmon resonance (SPR) sensors and tapping-mode atomic force microscopy (TM-AFM). Results show that both nanoscale surface chemical and structural properties of SAMs affect protein adsorption. Specifically, carboxylic acid-terminated SAMs formed at higher temperatures have more compact structures and generally promote protein adsorption, whereas the adsorbed amount of protein is similar on methyl-terminated SAMs regardless of solution temperatures at which SAMs are formed.