A biosensor matrix based on UV-initiated graft copolymerized poly(ethylene glycol) methacrylate and 2-hydroxyethyl methacrylate has been studied using imaging surface plasmon resonance (iSPR). By using a photo mask and a programmable shutter to vary the exposure time laterally, a gradient of matrix spots with physical thicknesses ranging from a few to tens of nanometers was generated. To maximize the dynamic range, imaging SPR was employed in wavelength interrogation mode. By finding the minimum in the reflectance spectra from each pixel of an image, SPR wavelength maps were constructed. The shift in SPR wavelength upon biospecific interaction was then measured both as a function of matrix thickness and composition. The performance of the matrix was evaluated in terms of immobilization of human serum albumin, biomolecular interaction with its antibody, and nonspecific binding of human fibrinogen. In addition, a low molecular weight interaction pair based on a synthetic polypeptide and calmodulin was also studied to explore the size selectivity of the hydrogel matrix. Our results show that the gradient matrix exhibits excellent properties for quick evaluation and screening of optimal hydrogel performance. The mixed hydrogel matrices display very low levels of nonspecific binding. It is also evident that the low molecular weight calmodulin is capable of freely diffusing and interacting throughout the entire hydrogel matrix, whereas the much larger albumin and its corresponding antibody, in particular, are partly/completely hindered from penetrating the interior of the matrix. This size-selectivity is attributed to a significant UV-initiated cross-linking or branching of the matrix during fabrication and/or protein mediated multipoint attachment during immobilization.