Reverse osmosis (RO) technology can get rid of small molecular contaminants and salts with a very high efficiency, so that it has exhibited promising potential for addressing the emerging problem of a clean water shortage. In this paper, one new thin film composite (TFC) membrane with a novel MWCNT/polyamide (MWCNT/PA) composite rejection layer had been manufactured via interfacial polymerization (IP) method under negative pressure. The MWCNTs framework was first fabricated on surface of PVDF substrate and then the polyamide matrix was filled into the framework by IP process between the two monomers (M-phenylenediamine and trimesoyl chloride) under negative pressure. The novel rejection layer obviously enhanced the water flux and antifouling performance without harming the desalination rate in RO process. The improved performances can be attributed to the larger amount of hydrophilic MWCNTs which were embedded into the rejection layer and worked as water channels. Meanwhile, this research also revealed that enhancing the density of water channels in polyamide rejection layer and the hydrophilicity of membrane surface could effectively improve the TFC membrane performance.