Various hydrogels have been used for protein delivery in the treatment of human diseases. Nevertheless, it is always difficult to control the capture and release of multiple proteins in different regions and periods. This research successfully proves that multiple proteins can be captured and released from the aptamer-patterned hydrogel films with an adjustable release rate at a prospective time and in specific regions utilizing the complementary DNA strand of aptamers via photoclick chemistry and DNA hybridization. The hydrogel film is successfully applied to complex matrixes such as human serum and has excellent cytocompatibility. Thus, the aptamer-patterned hydrogel film will be a good candidate for controlled delivery of multiple proteins.