Deoxyribozymes (DNAzymes) are single stranded DNA molecules exhibiting catalytic activity that can be exploited in medicine, biology and material sciences. DNAzymes with mimicking peroxidase activity enjoy particularly great interest because of their bioanalytical potential. In order to assure such a catalytic activity, the oligonucleotide has to adopt a G-quadruplex structure and specifically bind the prosthetic hemin molecule. This review presents fundamentals concerning design and engineering of DNAzymes with peroxidase-like activity, describes their properties and spectral characteristics and shows how DNAzymes contribute to bioanalytical research. Presented here examples of bioanalytical applications of DNAzymes with peroxidase-like activity include nucleic acid probes with DNAzyme labels for detection of specific DNA sequences in colorimetric or chemiluminescent assays. Telomerase or methyltransferase activity, which are potential targets in anticancer therapy, have also been studied. Other applications include determination of metal cations such as Ag⁺, K⁺, Hg²⁺, Pb²⁺, or Cu²⁺, amplified detection of small molecules (e.g., adenosine, cocaine, or AMP) as well as proteins (e.g., lysozyme or thrombin). Concluding, DNAzymes have made in last decade a great journey and brought numerous applications in many areas of science from chemistry through biology to medicine.