Owing to the high sensitivity, excellent programmability, and flexible obtainment through in vitro selection, RNA-cleaving DNAzymes have attracted increasing interest in developing DNAzyme-based sensors. In this review, we summarize the recent advances on DNAzyme-based sensing applications. We initially conclude two general strategies to expand the library of DNAzmes, in vitro selection to discover new DNAzymes towards different targets of interest and chemical modifications to endue the existing DNAzymes with new function or properties. We then discuss the recent applications of DNAzyme-based sensors for the detection of a variety of important biomoleucles both in vitro and in vivo. Finally, perspectives on the challenges and future directions in the development of DNAzyme-based sensors are provided.