Human telomerase RNA (hTR), a template of telomerase for telomeric repeat synthesis, was used to reflect the telomerase activity and act as a potential target of antitumor therapy. Here, we report a novel DNA-conjugated AuNP probe termed sticky-flares for the in situ detection of intracellular human telomerase RNA. The sticky-flares probe is capable of entering living cells directly without any auxiliary and recognizing the binding domain of human telomerase RNA. On recognition, the fluorophore-modified recognition flares can specifically bind to the target, separate from the sticky-flares and act as a fluorescent reporter to quantify and dynamically profile human telomerase RNA in living cells. We envision that the sticky-flares probe would be a valuable platform to investigate the function and regulation of hTR in antitumor therapy and hTR-related drug invention.