8-17 DNAzyme is a well-known versatile nucleic acid tool for achieving a specific cleavage function, and thus, investigation of 8-17 DNAzyme functions can prove to be of great significance. The conventional epigenetic modification on DNAzyme may pave a new way for the study of catalytic properties. Herein, the most abundant and best characterized modifications 5-methylcytosine (5mC) and N6-methyladenosine (m6A) are introduced into the central catalytic core and stem sequence of 8-17 DNAzyme to evaluate the cleavage activity. The modified 8-17 DNAzymes are arranged to recognize and cleave single-stranded DNA (ssDNA) substrates that contain a 5'-rAG-3' motif, producing large numbers of short ssDNA and leaving different amounts of undegraded ssDNA because of their disparate excision efficiency. Meanwhile, the undegraded ssDNA is used as new substrates for triggering the catalytic hairpin self-assembly (CHA) reaction. Benefiting from the facile and sensitive CHA reaction, the methylation-induced fluctuations of cleavage activity can be directly amplified and detected. Moreover, dioxygenase ten-eleven translocation protein 2 (Tet 2) offers a possibility for exploring the reversibility of methylation-modified DNAzymes through a demethylation process. In this study, we found that both 5mC and m6A modifications in the circular catalytic core might lead to a significant inhibition effect on the catalytic activity of 8-17 DNAzyme. However, little variation was observed when the stem region was labeled with 5mC. Additionally, the alkaline condition (pH = 9.5) enabled the partial recovery of cleavage activity for DNAzyme-19-5mC (∼52.9%). More impressively, these 8-17 DNAzymes were employed to study the regulation of miRNA-21 level in nonsmall cell lung cancer (A549) cells and human cervical cancer (HeLa) cells, revealing that the decrease of intracellular miRNA-21 content showed positive correlation with the death of tested cells. This study would hopefully advance the epigenetics research and dramatically expand the biosensing application of DNAzymes.