Maintaining colloidal stability in unfriendly environments while retaining surface chemical properties is challenging for fundamental science and crucial for many applications. Here, we report for the first time that by using a low concentration of poly(sodium 4-styrenesulfonate) (PSS), graphene-based amphiphilic Janus nanosheets (AJNs) can be stabilized in high salt brine (3 wt % NaCl and 0.5 wt % CaCl2), whereas the interfacial behavior of the nanosheets is not affected. The adsorption of PSS on the hydrophilic and hydrophobic surfaces of AJNs in brine was investigated experimentally and by molecular dynamics simulations. Simulations further showed that the spatial configuration of absorbed PSS molecules with sulfonate functional groups facing outward favored the generation of electrosteric repulsive interactions. Calculations of the interaction energy between PSS molecules and the nanosheet revealed surface charge as a key parameter to stabilize AJNs in the salt environment, as demonstrated by the case of graphene oxide with higher surface charge. Simulations were also used to examine the interfacial behavior of graphene-based AJNs in biphasic systems. The AJNs, which exhibited asymmetry in surface wettability, remained at the oil/brine interface because of PSS detachment from the hydrophobic surface. The results were subsequently experimentally confirmed, consistent with our previously reported graphene-based AJN fluid prepared in fresh water. The process was thermodynamically supported by the demonstrated negative change of Gibbs free energy. We believe that such a strategy could benefit for the stabilization of other AJNs with surface chemical accessibility under harsh conditions.