As a universal tumor biomarker, research on the activity and inhibition of telomerase is of great importance for cancer diagnosis and therapy. Herein, we demonstrate the conformational switch-based fluorescence detection of telomerase activity using a redesigned RNA aptamer Spinach. Briefly, the original Spinach aptamer was extended at its 5' end and folded into an inactive conformation, where association with the small molecule fluorophore, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) was prevented. Only in the presence of telomerase, (TTAGGG)n repeats were added to the 3' end of the telomerase substrate primer, and the elongation products hybridized with inactive Spinach molecules, triggering its conformational switch and refolding it into the active, DFHBI-binding conformation. Moreover, the fluorescence signal was further amplified through a target recycling circuit, where Ribonuclease H (RNase H) specifically hydrolyzed the phosphodiester bonds of RNA in the DNA-RNA hybrid. The released telomere products could then hybridize to new inactive Spinach molecules and initiate multiple amplification cycles. The proposed fluorescent biosensor presented great performance for telomerase activity detection from 100 to 5 × 104 Hela cells with a detection limit of 100 cells. Besides, this new assay offers a good biosensing platform for differentiation of cancer cell lines from normal cell line and evaluation the inhibition efficiency of telomere-binding ligand, which is of great importance for telomerase-related cancer diagnosis and therapy.