The polyamide reverse osmosis (RO) membranes were prepared through interfacial polymerization of m-phenylenediamine (MPD) and trimesoyl chloride (TMC). The use of dimethyl sulfoxide (DMSO) and glycerol as additives for the formation of thin-film composite (TFC) was investigated. We studied the effect of DMSO and glycerol addition on membrane property and RO performance. Microscopic morphology was examined by atomic force microscopy and scanning electron microscopy. The surface hydrophilicity was characterized on the basis of water contact angle and surface solid-liquid interfacial free energy (-ΔGSL). Water flux and salt rejection ability of the membranes prepared with or without the additives were evaluated by cross-flow RO tests. The results reveal that the addition of DMSO and glycerol strongly influences the property of the TFC RO membrane. Compared to the MPD/TMC membrane fabricated without DMSO and glycerol, the MPD/TMC/DMSO/glycerol membrane has a rougher surface and is more hydrophilic, showing smaller water contact angle and larger -ΔGSL value. Without decrease in salt rejection ability, the MPD/TMC/DMSO/glycerol membrane shows water flux significantly larger than that of the MPD/TMC membrane. The unique property of the MPD/TMC/DMSO/glycerol membrane is attributed to the cooperative effect of DMSO and glycerol on membrane structure during the interfacial polymerization process.