As an important biomarker for early cancer diagnosis and a valuable therapeutic target, the detection and monitoring of telomerase activity has attracted extensive attention. Herein, we constructed a novel fluorescent reporter to detect the intracellular telomerase activity based on guanine enhanced sliver nanoclusters (Ag NCs). In this system, a telomerase substrate (Ts) was designed to be extended by telomerase to give the repeat TTAGGG sequence as G-rich DNA, which was complementary to the nine base sequence to form a self-hairpin structure. The proximity of G-rich DNA to the as-prepared Ag NCs resulted in a 13-fold enhancement of fluorescence intensity due to the electron transfer from guanine to the Ag NCs. Therefore, the fluorescent probe could realize the detection of telomerase activity. Furthermore, the probe was successfully used to distinguish normal cells from cancer cells and to monitor the real time telomerase activity response upon treatment with an inhibiting model drug. This detection technique is simple, with excellent biocompatibility, inexpensive and expands the applications of the Ag NC probe.