A new reversible addition-fragmentation chain transfer (RAFT) agent, biotinylated trithiocarbonate, was successfully synthesized. Temperature-responsive biotinylated polymer, poly( N-isopropylacrylamide) (PNIPAAm) and water-soluble poly( N-(2-hydroxypropyl) methacrylamide) (PHPMA), have been prepared in one-step via RAFT polymerization using biotinylated trithiocarbonate as the RAFT agent. The results show that the polymerizations of N-isopropylacrylamide (NIPAAm) and N-(2-hydroxypropyl) methacrylamide ( HPMA) are of living characters, and the corresponding polymers produced have ending biotin group. Biotin-ended diblock copolymer of PHPMA and PNIPAAm was also prepared by RAFT polymerization of NIPAAm using biotinylated PHPMA as macro chain transfer agent. The biotinylated PHPMA-b-PNIPAAm can form a new kind of core-shell nanostructure with biotin groups on the surface reversibly induced by temperature.