Hydrogel, a water-filled network that can adapt to external stimuli by altering its volume, is known for its high flexibility and biocompatibility. DNA, a critical biomolecule renowned for its exceptional characteristics including information transmission, molecular recognition, and editability, has found widespread applications in biosensing field as well. The integration of these two biomaterials offers promising opportunities for the development of novel biosensors with enhanced sensitivity, specificity, and adaptability. Therefore, by virtue of the collective features, researchers have recently focused on the construction of responsive DNA hydrogel systems. This feature article describes recent development on fabricating DNA hydrogels and their applications in biosensing area. Initially, it focuses on the design strategies employed in preparing DNA hydrogels, encompassing both pure DNA hydrogels and hybridized DNA hydrogels. Subsequently, it summarizes the use of DNA hydrogels in biosensing applications, highlighting their applications in visual detection, electrochemical sensing, and optical analysis. Furthermore, the underlying responsive mechanisms within these biosensing systems are also described. Lastly, this article presents a comprehensive discussion on the existing challenges and prospects of responsive DNA hydrogels, offering insights into their potential to revolutionize the field of biosensing.