alpha-Glutathione (GSH), omega-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 H-1 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 alpha-allyl, omega-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 alpha-GSH, omega-biotin functionalized PNIPAAm. Glutathione S-transferase (GST) and streptavidin (SAv) were coupled in solution with heterofunctional PNIPAAm via bioaffinity interactions. Separately, alpha-GSH, omega-biotin functionalized PNIPAAm was further shown to bind GST-tagged Rac1, a potential cancer marker, and biotin-tagged bovine serum albumin (BSA).