Amphiphilic nanosheets have gained great attraction in diverse applications owing to their unprecedented performance. In this paper, amphiphilic molybdenum disulfide (CTAB-MoS2) nanosheets were prepared through a one-step simple hydrothermal method, and the physicochemical properties were systematically investigated. The results showed that the CTAB-MoS2 nanosheets displayed a distinct ultrathin flakelike structure and a lateral size in the range of 95 nm. More specifically, the presence of defect sites over the surfaces of MoS2 nanosheets facilitated linkage with CTAB molecules, which made MoS2 amphiphilic. Because of the amphiphilic nature, the CTAB-MoS2 nanosheets could be steadily dispersed in water phase, reduce the oil-water interfacial tension, alter wettability of the solid surface, and stabilize emulsions at ultralow concentrations (50 mg/L). By observing and analyzing the interfacial behavior of CTAB-MoS2 nanosheets in a simulated oil/brine system, it was found that CTAB-MoS2 nanosheets could be adsorbed at the oil-water interface in the form of multilayer adsorption, and the adsorption layer (oil-water interfacial film) exhibited higher strength elasticity. This work provides a scalable method for preparing amphiphilic nanosheets and reveals the physicochemical properties of CTAB-MoS2 amphiphilic nanosheets.