DNA hydrogels have emerged as a focal point in biomaterials research, attracting significant interest owing to their distinctive characteristics. These hydrogels are programmable, biocompatible, and possess an exceptional ability to form complex structures within physiological environments. This review offers a thorough overview of the recent progress and applications of DNA hydrogels across various domains, including tissue engineering, drug delivery, medical imaging, cell culture, wound dressing, and bioprinting. In the realm of tissue engineering, DNA hydrogels are utilized as versatile scaffolds that closely simulate the extracellular matrix. This similarity not only enhances cell adhesion but also promotes cell proliferation, thereby facilitating tissue regeneration. When it comes to drug delivery, the adjustable porosity of these hydrogels allows for a controlled release of therapeutic agents, significantly improving their efficacy and targeting capabilities. Furthermore, the integration of imaging agents into DNA hydrogels enhances the visibility of biological structures, providing a powerful tool for more accurate diagnostics. In cell culture applications, DNA hydrogels offer cutting-edge platforms that support various aspects of cell behavior and differentiation, fostering a better understanding of cellular processes. As for wound dressings, these hydrogels maintain a moist environment that is conducive to healing and possess inherent antimicrobial properties, making them particularly effective for managing chronic wounds. Moreover, DNA hydrogels facilitate the bioprinting of complex three-dimensional tissue constructs, ensuring an accurate spatial arrangement of cellular components that is crucial for functional tissue development. This review consolidates the existing knowledge surrounding DNA hydrogels, highlighting their multifaceted roles in biomedical applications. It underscores the potential of these innovative materials to transform the fields of regenerative medicine and drug delivery.