A method of fabricating photo-cross-linked hydrogels with gradients of immobilized molecules and cross-linking densities is introduced. Two macromer/initiator solutions are injected into a unique poly(dimethylsiloxane) channel system that produces a prepolymer gradient that is subsequently polymerized into a water-swollen hydrogel with ultraviolet light exposure. The gradient is controlled by the injection flow rate (optimized to 0.3 muL/min per inlet to produce a linear gradient). The technique is investigated both through fabrication of adhesive ligand gradients that modulate spatial distribution of attached endothelial cells and gradients of cross-linking densities that led to unique hydrogel architectures and spatially dependent swelling.