Hydrophobic agents, a fluorescent dye (Nile red, NR) or an anticancer drug (doxorubicin, DOX), are encapsulated into poly((N-[3-(dimethylamino) propyl] methacrylamide)-b-poly (methyl methacrylate) (PDMAPMA-b-PMMA) nanoparticles (NPs) via one-pot reversible addition-fragmentation chain-transfer (RAFT)-mediated emulsion polymerization in water. The macroRAFT, PDMAPMA, is chain-extended with the methyl methacrylate (MMA), with the hydrophobic agents soluble in MMA, resulting in loaded NPs, with either NR or DOX via polymerization-induced self-assembly (PISA). The NR-loaded NPs are visualized by structured illumination microscopy (SIM), thus indicating the successful loading of the fluorescent dye into the PMMA core. The DOX-loaded NPs exhibit a sustained release profile over 5 d, showing a small burst effect during the first 2 h, as compared with the free DOX. The DOX-loaded NPs show higher cell toxicity than the free DOX in RAW 264.7 cell line. The results demonstrate the potential of using emulsion polymerization for synthesis of tailored and reproducible NPs encapsulating hydrophobic agents.