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Telomerase is considered a valuable diagnostic and prognostic cancer biomarker. Accurate and reliable detection of telomerase activity is of great value in clinical diagnosis, screening of inhibitors, and therapeutics. Here, we developed a novel amplified fluorescence resonance energy transfer (FRET) nanoprobe for highly sensitive and reliable monitoring of intracellular telomerase activity. The nanoprobe (QDSA@DNA) was composed of a streptavidin-modified quantum dot (QDSA) which was functionalized with a telomerase primer sequence (TP) and Cy5-tagged signal switching sequence (SS) through biotin-streptavidin interaction. When the nanoprobe was assembled, the Cy5 was in close proximity to the QDSA, resulting in high FRET efficiency from the QDSA to Cy5. In the presence of telomerase, the TP could be extended to produce telomeric repeat units, which was complementary to the loop of SS. Thus, the SS could hybridize with elongated sequences to form a rigid double-stranded structure, which forced the Cy5 away from the surface of the QDSA, causing low FRET efficiency. Furthermore, due to the production of multiple repeat units by telomerase, multiple hairpin structures could be opened, yielding significant fluorescence ratio (FQDsa/FCy5) enhancement for sensing of telomerase activity. In this way, the combination of a FRET and target-assisted strategy in a nanoprobe improved the detection accuracy and amplified the detection signal, respectively. The QDSA@DNA nanoprobe also showed high selectivity, excellent nuclease stability, and good biocompatibility. More importantly, this nanoprobe was found to be an excellent platform for efficient monitoring of intracellular telomerase activity, providing a potential platform in tumor diagnosis and screening of telomerase-related inhibitors. [ABSTRACT FROM AUTHOR]