An amphiphilic asymmetric comb polymer with pendant pyrene groups and poly(N-isopropylacrylamide) (PNIPAM) side chains was synthesized based on click chemistry and reversible addition-fragmentation chain transfer polymerization. Gel permeation chromatography, FTIR, and H-1 NMR results all indicated successful synthesis of a well-defined comb polymer. The photophysical properties and self-assembly of the polymer in solution were studied by UV-vis spectroscopy, fluorescence technique, and transmission electron microscopy. The intensity ratio of the excimer peak (I-E) to the monomer peak (I-M) of the comb polymer in THF was used to monitor the formation of inter- or intramolecular excimers. At low polymer concentration, the value of I-E/I-M kept unchanged, indicating the formation of intramolecular excimer; at high polymer concentration, the value increased rapidly with concentration because of the formation of intermolecular excimer. The change of the intensity ratio of the first to the third vibronic band (I-1/I-3) on the monomer emission of the comb polymer also proved the association of the pendant pyrene groups in THF at high polymer concentration. In aqueous solution, the comb polymer chains self-assembled into vesicles with pyrene groups in the walls and PNIPAM side chains in the coronae. The value of the critical aggregation concentration of the polymer was determined by fluorescence technique. Temperature exerted a significant effect on the size and morphology of the vesicles. At a temperature above the lower critical solution temperature (LCST) of PNIPAM, PNIPAM brushes in the coronae of vesicles collapsed on the surface of the structures forming nanosized domains, and vesicles with smaller size were obtained. Fluorescence quenching experiments indicated that the collapsed PNIPAM chains protected a part of pyrene groups from being quenched by nitromethane at a temperature above the LCST of PNIPAM.