α-Glutathione (GSH), ω-biotin functionalized poly(N-isopropylacrylamide) (PNIPAAm) was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization using a new R-group allyl functionalized trithiocarbonate chain transfer agent (CTA) and thiol-ene reactions. GPC and 1H NMR results indicated that the allyl group had no adverse effect on the RAFT-controlled polymerization of NIPAAm and PEG-A, and the new CTA could efficiently control the polymerizations. Employing radical thiol-ene and Michael addition reactions, heterotelechelic α-allyl, ω-carboxylic acid-PNIPAAm was first aminolyzed in the presence of maleimide-modified biotin and subsequently reacted with GSH via radical thiol-ene addition to yield α-GSH, ω-biotin functionalized PNIPAAm. Glutathione S-transferase (GST) and streptavidin (SAv) were coupled in solution with heterofunctional PNIPAAm via bioaffinity interactions. Separately, α-GSH, ω-biotin functionalized PNIPAAm was further shown to bind GST-tagged Rac1, a potential cancer marker, and biotin-tagged bovine serum albumin (BSA).