Hydrogels are water-retainable materials that can absorb a large amount of water. Different stimuli can be used to stimulate the hydrogels with a variety of physical and chemical changes, thus leading to numerous applications in bioanalysis and biomedicine. Aptamers are special types of single-stranded DNA generated by a process called systematic evolution of ligands by exponential enrichment (SELEX). They are able to specifically recognize a wide range of targets which vary from ions, small molecules, to proteins, and even whole cells. Aptamer incorporation has greatly expanded the applications of hydrogels. Due to their unique properties, such as biocompatibility, selective binding, and molecular recognition, these aptamer-based hydrogels can be utilized for target-responsive hydrogel engineering. In this chapter, we discuss a variety of applications of aptamer-based hydrogels, especially in sensing, target capture and separation, control of target release and in vivo applications.