Therapeutic DNAzymes have unceasingly intrigued the scientific community owing to their prosperous gene regulation capability. The efficacy of DNAzymes against many types of diseases has been extensively studied for over two decades. However, the high expectations for DNAzymes are still not translated to the clinic because of their low effectiveness in vivo. Over the last five years, several aspects have been considered to optimize DNAzyme-integrated therapeutics, including structural stability, mechanism exploration, cell internalization rate, cofactor activation, and off-target effects. Hence, this review first discusses the early monotherapy and structural design of DNAzymes. Subsequently, the latest modes of action are reviewed, followed by an elaboration on structural stabilization strategies considering the catalytic core and substrate-binding arms. DNAzyme-based synergistic therapy is then examined, highlighting responsive carrier construction, synergistic effects, the latest discovered advanced functions, and off-target concerns. Beyond this, key clinical advances in DNAzyme-based therapy are elucidated by showcasing clinical progress. Finally, future trends and development challenges for DNAzyme-powered therapeutics in the coming years are discussed in detail.