Stimulus-responsive hydrogels, with biocompatibility, sufficient water content, similarity to extracellular matrices, and responses to specific environmental stimuli, have recently received massive research interest for fabricating bioactuators. The potential of employing these hydrogels that respond to various stimuli (e.g., pH, temperature, light, electricity, and magnetic fields) for actuation purposes has been uncovered by their performances in biosensing, drug delivery, artificial muscle reconstruction, and cell microenvironment engineering. In this review, a material selection of stimulus-responsive hydrogels and a detailed discussion of recent advances in emerging biomedical applications of hydrogel-based bioactuators are proposed. Existing challenges and future prospects are noted as well.