Azido-containing amphiphilic triblock copolymer poly(ethylene glycol)-b-poly(azidoethyl methacrylate)-b-poly(methyl methacrylate) (PEG-b-PAzEMA-b-PMMA) was prepared by postpolymerization functionalization of poly(ethylene glycol)-b-poly(hydroxyethyl methacrylate)-b-poly(methyl methacrylate) (PEG-b-PHEMA-b-PMMA). In aqueous media, PEG-b-PAzEMA-b-PMMA self-assembled into spherical micelles with the azide groups at the hydrophobic/hydrophilic interface due to the molecular architecture. Biotin was conjugated to the micelles by in situ click chemistry between azide groups and alkynated biotin, resulting in the formation of a functional interface between the hydrophilic shell and the hydrophobic core. The bioavailability of biotin to avidin was demonstrated by an avidin/4'-hydroxyazobenzene-2-carboxylic acid (avidin/HABA) assay, transmission electron microscopy, and dynamic light scattering investigations.