Biomolecular patterning is essential for the creation of sensing motifs that rely on receptor-ligand binding for selectivity. Microfluidic devices have the potential to aid in the development of simple, robust methods for biomolecular patterning and therefore contribute to the generation of protein, DNA, and cell microarrays. In microfluidic patterning, the choice of both substrate and microfluidic channel material is essential for control of both the receptor binding for maximal signal generation as well as non-specific adsorption that acts as chemical noise. In this study, polystyrene, glass, silicon nitride, and poly(dimethylsiloxane) (PDMS) were evaluated as substrates for protein patterning using two types of PDMS microchannels for patterning, native PDMS and solvent-extracted PDMS (E-PDMS). E-PDMS microfluidic channels resulted in better patterning characteristics than native PDMS channels as determined by a higher fluorescence intensity of immobilized protein on all substrate types tested. Microfluidic patterning was then applied to perform two- and four-layer immunoassays.