We describe the preparation of hydrogels using highly functionalized poly(oxazoline) based polymeric precursors and cross-linking via UV mediated radical thiol-ene chemistry. Random copolymers were synthesized based on the combination of the more hydrophilic 2-methyl-2-oxazoline or the less hydrophilic monomer 2-ethyl-2-oxazoline with 2-(3-butenyl)-2-oxazoline. These copolymers were functionalized via a post-polymerization technique with thiol or cysteine functionality at the side chain. Hence, hydrogels were obtained, for which the thermo-responsive behavior, network density and correlated properties such as swelling and mechanics, as well as the possibility of electrostatic interaction, can be tuned. Cell culture tests demonstrated good cytocompatibility of the synthesized copolymers and hydrogels. A study with two low molecular weight substances, methylene blue and fluorescein sodium, was performed to investigate how the thermo-responsive behavior or the positive charge incorporated by cysteine could influence the interaction with the compounds. It was found that the interaction with the hydrogel network was strongly influenced by the chemical properties of the dye. A hydrophilic and positively charged hydrogel network was shown to be a promising candidate for the uptake and prolonged release of negatively charged low molecular weight substances.