Thiolactone-functionalized polyurethanes were produced by using a chain extension method. Isocyanate-telechelic urethane prepolymers of different chain lengths were prepared from two different diols and 4,4'-methylenebis(cyclohexyl isocyanate), and then reacted with two different thiolactone-functionalized diols, yielding polyurethanes bearing thiolactone groups either within or along the polymer backbone. Postmodification reactions were performed according to an amine-thiol-thiosulfonate conjugation strategy using various functional amines and thiosulfonates. Different characterization techniques were implemented to prove the double modification, for example, H-1 NMR, F-19 NMR, SEC, DSC, and fluorometry.