Two dimensional (2 D) layered materials with special properties provide opportunities to design energy-efficient membranes for water desalination. A negatively charged layered graphene analogue, molybdenum disulfide (MoS₂) was synthesized through liquid exfoliation. A novel thin film nanocomposite (TFN) reverse osmosis (RO) membrane was successfully fabricated with prepared laminar MoS₂ as fillers in the organic phase through interfacial polymerization of m-phenylenediamine and trimesoyl chloride monomers. The laminar MoS₂ nanosheets were observed by transmission electron microscopy (TEM), and the nanosheets were dispersed in the polyamide (PA) matrix as well as on the membrane surface. The properties of the resultant TFN membranes were effectively modified by taking the advantage of two dimensional features, i.e., the negative charge and the hydrophilic sites of layered MoS₂. Compared to a classical TFC membrane, the MoS₂ incorporated TFN membranes were found to have an increased salt rejection and higher water flux. Moreover, 0.01 wt% MoS₂-TFN membrane showed a superior fouling resistance against proteins as foulants. The antifouling behavior of this TFN membrane was observed by field-emission scanning electron microscopy (FE-SEM). Stability tests demonstrated that there was a loss of MoS₂ in the initial 2 h filtration, but the process is stable in the long-term.