Human telomerase is a ribonucleoprotein complex which adds the repeats of TTAGGG on the telomeric ends of chromosomal DNA. Even though the vast majority of human cancers express telomerase activity, most human somatic cells lack the telomerase activity; consequently, telomerase has been regarded as both a biomarker for early cancer diagnosis and a therapeutic target. Here we develop a simple, rapid and highly sensitive method for the detection of telomerase activity at the single-cell level using telomeres-induced two-stage isothermal amplification-mediated chemiluminescence assay. In the presence of telomerase, the telomere repeats of (TTAGGG)n are added to the 3' end of telomerase substrate primer, which can be converted to the template of strand displacement amplification (SDA) for the generation of short oligonucleotides, catalytic DNAzymes, and the telomere repeats of (TTAGGG)n. The short oligonucleotide might function as a new trigger to bind the free telomerase substrate primer and consequently initiate an isothermally exponential amplification reaction (EXPAR), generating a large number of catalytic DNAzymes. Both the DNAzymes and the G-rich telomeric repeat units can interact with hemin to form the catalytic hemin-G-quadruplex nanostructures, which can catalyze the generation of amplified chemiluminescence signals in the presence of luminol and H2O2. While in the absence of telomerase, the two-stage isothermal amplification cannot be initiated, and no chemiluminescence signal is observed. Owing to the high amplification efficiency of two-stage isothermal amplification as well as the high sensitivity and wide dynamic range of the chemiluminescence assay, the proposed method can sensitively measure the synthetic telomerase product TPC8 with a detection limit of as low as 0.1 aM and a large dynamic range of 10 orders of magnitude from 0.1 aM to 1 nM and can even detect the telomerase activity from a single HeLa cancer cell without the need for any labeled DNA probes. The proposed method can be further used to screen the anticancer drugs and might provide a promising approach for the discovery of new anticancer drugs.