The invention relates to a surface-enhanced Raman technology based on signal-off and used for detecting intracellular telomerase activity. According to the technology, graphene, carbon nitride, MoS2, SiO2 and the like are taken as carriers, nano-gole or nano-silver with controllable dimension and morphology or a composite magnetic nano-particle with a core-shell structure is supported by the carriers, and then a hairpin probe containing a telomerase primer and a Raman molecule beacon is fixedly disposed on the nano=particle surface, so that the high-sensitivity high-selectivity SERS detection method based on signal-off is established. When a target exists, the primer extends and generates a DNA chain with a repeat sequence under the effect of telomerase, and the DNA chain is hybridized with the probe molecule, then the DNA hairpin structure is opened, a Raman signal molecule with the single-chain labeled end is far away from a Raman substrate, the Raman signal is reduced, and high-sensitivity detection on telomerase is realized. The probe prepared by taking Hela cervical carcinoma cell as a model realizes intracellular telomerase activity detection and dynamic detection on intracellular telomerase activity variation under effect of a telomerase inhibiting medicament. By using multiple cells for imaging, the probe is confirmed to be capable of distinguishing tumor cells and normal cells.