Poly(ethylene glycol)-based hydrogels containing thiol-reactive maleimide functional groups is prepared via a Diels-Alder/retro Diels-Alder reaction sequence using a masked maleimide monomer. Bulk and micropatterned hydrogels containing varying amounts of the thiol-reactive maleimide functional group are fabricated at ambient temperature. During the fabrication, the reactive maleimide functional group in the monomer is masked with a furan moiety and then unmasked to its reactive form via the retro-Diels-Alder reaction. The reactive maleimide groups embedded within the hydrogel are amenable to facile and efficient functionalization with thiol-containing molecules such as fluorescent dyes. Furthermore, these hydrogels are readily biotinylated using the nucleophilic thiol-ene conjugation to enable immobilization of streptavidin onto the hydrogel patterns to achieve facile bioimmobilization. Notably, the extent of functionalization of these hydrogels can be easily tailored by varying the amount of reactive handles incorporated during their fabrication.