We developed a three-dimensional polyglycerol-polyethylenglycol based hydrogel as a new biosensing matrix for affinity analysis by surface plasmon resonance to enable a higher loading of ligand for small molecule analysis. The hydrogel was synthetized by crosslinking a polyglycerol functionalized with carboxylate and maleimide groups with a dithiolated polyethyleneglycol by thiol-click chemistry. We demonstrated that the hydrogel coating enabled a high immobilization capacity of biomolecules and thus signal amplification of the binding response. Here, the degree of loading with carbonic anhydrase II and the resulting binding signal of acetazolamide were increased by factor 5 compared to standard CMD sensors (CM5) and the loading was comparable to CMD sensors specialized for maximum loading (CM7). This high loading ability combined with the reduced nonspecific binding due to the missing carbohydrate structure presents an innovative matrix for a broad application range of SPR experiments.