DNA glycosylase dysregulation is implicated in carcinogenesis and therapeutic resistance of cancers.Thus, various DNA-based detection platforms have been developed by leveraging the base excision activity of DNA glycosylases.However, the efficacy of DNA-based methods is hampered due to nonspecific degradation by nucleases commonly present in cancer cells and during preparations of cell lysate.In this report, we describe a fluorescence-based assay using a specific and nuclease-resistant three-dimensional DNAzyme walker to investigate the activity of DNA glycosylases from cancer cell lysates.We focus on DNA glycosylases that excise uracil from deoxyuridine (dU) lesions, namely uracil DNA glycosylase (UDG) and single-stranded monofunctional uracil DNA glycosylase (SMUG1).Limits of detection for detecting UDG and SMUG1 in buffer were 3.2 pM and 3.0 pM, resp.The DNAzyme walker detected uracil-excision activity in diluted cancer cell lysate from as low as 48 A549 cells.of the UDG inhibitor experiments demonstrate that UDG is the predominant uracil-excising glycosylase in A549 cells.Approx. 500 μM of UDG is present in each A549 cell on averageNo fluorescence was generated in the samples lacking DNAzyme activation, indicating there was no nonspecific nuclease interference.The ability for the DNAzyme walker to respond to glycosylase activity illustrates the potential use for DNAzyme walker technol. to monitor and study biochem. processes involving glycosylases.