A series of α-biotinylated poly(N-isopropylacrylamide) (PNIPAAm) block copolymers with different poly(ethylene glycol) (PEG) spacers were synthesized and two approaches for ω-functionalization were tested, first, by the use of atom transfer radical polymerization (ATRP) and sequential monomer addition and second, by halogen end group modification with azides and subsequent copper catalyzed azide–alkyne cycloadditions. Three different dyes, rhodamine B, fluorescein and a coumarin derivative, were chosen as end groups. For each approach the corresponding monomer or alkyne functionalized dye was synthesized. The biotin group can be used for attaching the polymers to streptavidin whereas the fluorescent dyes can be used as labels for polymer monitoring in various environments and thus to larger structures (e.g. microtubule, surfaces, etc.). The synthesis routes of the biotin–PEG initiators were developed in a way that also amino functionalized PEG initiators as intermediates were produced. The advantage of this approach is the use of these polymers for other purposes like direct covalent coupling to biomolecules, fluorescent labeling or surface attachment, gaining a very flexible tool for PNIPAAm functionalization. The two approaches for ω-functionalization were investigated by NMR, GPC and UV-Vis spectroscopy and compared for their efficiency.