Telomerase is an important reverse transcriptase that is in charge of maintaining telomeres length, and the dysregulation of telomerase activity is closely associated with cancers. Accurate measurement of telomerase activity is critical to clinical diagnosis and therapeutics. Herein, by taking advantage of in vivo repair mechanisms and single-molecule detection, we demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells. The presence of telomerase enables repetitive addition of (TTAGGG)n to the 3′-OH end of telomerase substrate (TS) primer to generate long telomeric products. Telomeric product then hybridizes with the apurinic (AP) probe to induce the cleavage of AP site in dsDNA by APE1, yielding a ssDNA with a biotin and an OH group at 5′ and 3′ ends and simultaneously releasing the telomeric product which in turn hybridizes with new AP probes to yield numerous ssDNAs. By adding streptavidin-coated MBs, the ssDNAs are captured and subsequently functioned as primers to initiate template-free TdT-catalyzed random incorporation of dATPs and Cy5-dATPs into 3′-OH ends, producing long poly-A chains with multiple Cy5 molecules encoding and finally forming Cy5-ssDNA-MB complexes. After magnetic separation, Cy5-ssDNA-MBs are digested by Exo I, releasing large numbers of Cy5 molecules which can be measured by single-molecule detection. This strategy exhibits excellent specificity and high sensitivity with a limit of detection (LOD) of 1 cancer cell. Moreover, it can be employed to screen inhibitors and discriminate different cancer cell lines from normal cell lines, holding enormous potential in clinical diagnosis and therapeutics.