Although the preparation and application of amphiphilic Janus nanosheets have been studied a lot, little is known about their colloidal properties to date, especially at the molecular level. In this work, we systematically investigated the microscopic interactions between amphiphilic Janus nanosheets with different surface chemistry and water molecules. It is shown that the introduction of functional groups has a significant influence on the occupied volume and available surface area of the Janus nanosheets in aqueous solution due to the two-dimensional nature. The hydrophobic functional groups force water molecules away from the surface of nanosheets by forming a steric barrier. In contrast, the deprotonated hydrophilic groups have a strong and directional binding to the surrounding water molecules to form structured water layers on the surface of nanosheets. The interaction energy between Janus nanosheets and water molecules is dominated by electrostatic interaction and exhibit a strong dependence on the degrees of alkyl modification, the length of alkyl chain, and the types of hydrophilic groups on the surface of Janus nanosheets. The order of the influence of hydrophilic groups on the interaction energy and hydrogen bonding between Janus nanosheets and water is COO- > SO4- > COOH > NH2 > OH at 300 K. Moreover, the temperature and cation concentration of the aqueous solution have a significant effect on the interaction between water and Janus nanosheets with deprotonated hydrophilic groups. These results not only improve the understanding of the colloidal behaviors of the amphiphilic Janus nanosheets in aqueous solution but also contribute to the development of novel Janus materials for engineering applications.