Two well-defined amphiphilic asymmetric macromolecular brushes, one bearing hydrophilic poly(ethylene glycol) (PEO) and hydrophobic polystyrene (PS) side chains on poly(glycidyl methacrylate) (PGMA) backbone and the other bearing pendant PEO and poly(styrene-block-N-isopropylacrylamide) (PS-b-PNIPAM) block copolymer side chains, were synthesized by grafting from approach based on a combination of click chemistry and in situ reversible addition-fragmentation chain transfer (RAFT) polymerization. PGMA backbone was synthesized by atom transfer radical polymerization (ATRP), and a polymer backbone with pendant hydroxyl and azide groups (PGMA-OH/N-3) was obtained after ring-opening reaction of the epoxide rings on PGMA. RAFT chain transfer agent (CTA) was introduced to the polymer backbone by facile click reaction between alkyne-terminated RAFT CTA and PGMA-OH/N-3. PEO side chains were grafted onto the polymer backbone by esterification between carboxyl end group of PEO and hydroxyl group on the polymer backbone; PS or PS-b-PNIPAM side chains were prepared by RAFT polymerization. Gel permeation chromatograph, FTIR and H-1 NMR results all indicated successful synthesis of well-defined amphiphilic asymmetric macromolecular brushes. The self-assembly of the macromolecular brushes in solutions was also investigated in this research. Asymmetric macromolecular brushes with PEO and PS side chains self-assembled into vesicle structures in methanol. PS side chains were in the walls of the vesicles, and PEO side chains were in the coronae. The average size of the structure increased with PS chain length. The macromolecular brushes with PEO and PS-b-PNIPAM block copolymer side chains were able to self-assemble into vesicles in aqueous solution. Temperature exerted a significant effect on the morphology of the structures. At a temperature above lower critical solution temperature (LCST) of PNIPAM, the size of the vesicles decreased due to the shrinking of PNIPAM blocks in the corona.