DNA catalysts (Deoxyribozymes, DNA enzymes; DNAzymes) are catalytic artificial single-stranded DNA molecules. Using the technique of in vitro selection, individual DNA catalysts have been identified that catalyze RNA cleavage, RNA ligation and a wide range of other chemical reactions. DNA catalysts have been used in vitro as biochemical tools, analytical tools and sensors. DNA catalysts have also been utilized as in vivo therapeutic agents to target specific mRNA. Although many conceptual and practical challenges remain to be addressed, DNA catalysts make new promise for applications both in vitro and in vivo. In this review, we are focusing on definition and different types of DNA catalysts, in vitro Selection of DNA catalysts and their therapeutic applications. This review also describes the challenges and approaches to overcome obstacles involved in using DNA catalysts in the treatment of different diseases including cancers, viral and bacterial infection.