A robust method for the synthesis of the well-defined polyurethane-graft-poly(N,N-dimethylacrylamide) (PU-g-PDMA) copolymers with a good control over the graft density and the grafted chain length was presented in this study. Firstly, functional polyurethane (fPU) polymer with trithiocarbonate-based chain transfer agent lateral group was synthesized by the polyaddition reaction of 2,2-bis(hydroxymethyl)butyl 2-(ethylthiocarbonothioylthio)-2-methylpropanoate (BEMP) with hexamethylene diisocyanate (HDI), and the subsequent chain extension reaction with 1,4-butanediol (BDO). The content of the chain-transfer groups in the synthesized fPU could be well tuned by altering the mole ratio of BEMP to HDI during the synthesis of prepolymer. The produced fPU was then applied as the macro-RAFT to mediate the radical polymerization reaction of the N,N-dimethylacrylamide (DMA) monomers that were initiated by azobisisobutyronitrile (AIBN), resulting in well-defined amphiphilic PU-g-PDMA graft copolymers with a known graft density and tunable grafted chain lengths. The structure of the obtained PU-g-PDMA was characterized carefully by FTIR and 1 H NMR. The average molecular weight and polydistribution of PU-g-PDMA copolymers were analyzed by GPC. The thermal properties of fPU and PU-g-PDMA copolymers were investigated by differential scanning calorimetry (DSC) and themogravimetric analysis (TGA). The amphiphilic PU-g-PDMA graft copolymers could self assemble into spherical nanoparticles with a core-shell structure in water. The core-shell structure nanoparticles could be applied as emulsifiers for the formation of stabilized toluene in water Pickering emulsion, and an extremely low content of emulsifiers (~0.01%) relative to the total weight of oil and water was required.