Graphene oxide (GO) has been extensively applied in environmental and chemical engineering applications, such as wastewater remediation and organic compounds sensing, in which the adsorption of aromatic organic compounds (ADCs) (e.g., dyes, drugs and pesticides) on GO is commonly involved. Understanding the fundamental interaction mechanism of AOCs-GO is critical for these applications and designing advanced functional GO-based nanocomposites. In this work, for the first time, the nanomechanical interaction mechanism between GO and a model AOC molecule is quantitatively characterized using single-molecule force spectroscopy (SMFS) and density functional theory (DFT) simulations. The contributions of major functional groups of GO to the binding affinity and configurations of AOC/GO complex have been investigated. It is found that most binding events in the formation of AOC/GO complex should be attributed to the attraction between the cationic AOC molecule and the regions on GO with epoxy groups, the role of which has been overlooked previously, with an activation Gibbs energy for bond dissociation (Delta G) of -4.61 kcal mol(-1). Surprisingly limited occurrence of binding events between the cationic AOC and the regions with ionized carboxylic groups on GO suggests that the role of electrostatic interaction has been overrated traditionally. Our results provide new nanomechanical insights into the interaction mechanism of AOCs-GO with useful implications for developing novel AOC/GO nanocomposites with environmental, biomedical and engineering applications.