Supercritical carbon dioxide (ScCO2) fracturing technology has been increasingly emphasized in the development of unconventional oil and gas resources due to its advantages of anhydrous fabrication of complex seam networks and geological sequestration of CO2. However, previous studies have mostly focused on the geometry, number, and type of inorganic mineral fractures, and rarely paid attention to the changes in the seepage structure within organic matter (OM). To characterize the nanoscale microstructural changes of OM induced by H2O-ScCO2, this study obtained the variation rule of mechanical properties of OM with adsorbed water saturation based on nanoindentation test, and the continuous change of elastic modulus and morphology of the OM in the same region after dry, water-wet, and water-ScCO2 treatments was compared by atomic force microscopy (AFM). Elastic modulus and hardness of OM were calculated by Oliver-Pharr and Hertz methods, and the average roughness, root mean square roughness, and kurtosis were obtained based on 3D morphology. The results showed that elastic modulus and hardness were negatively and nonlinearly correlated with water saturation. Although the AFM-measured elastic modulus of OM microphases of the dry, water-wet, and water-ScCO2-treated shale were all highly discrete and non-homogeneous, they were generally normally distributed, and the mean values of the elastic modulus decreased sequentially. It is determined that water-ScCO2 coupling make OM matrix undergo superimposed dissolution. It is shown that ScCO2 and water synergistically change the microstructure of shale OM, and the weakening of mechanical properties and the increase of surface roughness are very favorable for geological sequestration and long-term storage of CO2. •Young's modulus and hardness of organic matter showed a nonlinear negative correlation with water content.•Young's modulus and morphological changes of organic matter were analyzed at the nanoscale by in-situ AFM.•Water swelling decreased the roughness and Young's modulus of the organic matter.•The coupling of water and ScCO2 complicated the morphology and decreased Young's modulus of the organic matter.