The detection of telomerase activity inside cells is valuable for early cancer diagnosis and telomerase function study. However, besides cancerous cells, telomerase is also found to be expressed in few non-cancerous cells, which influences the assay reliability. By virtue of the extracellular pH, we design a DNA tetrahedron docking assembly (DTDA) for only responding telomerase activity in cancerous cells. The DTDA maintains structural integrity with extracellular acid pH of cancerous cells, but releases a telomerase substrate-containing strand after its cell internalization due to intracellular alkaline pH. The strand gets elongated by intracellular telomerase, docks to the vertex of tetrahedron, and returns to the DTDA after its separation, accompanied by fluorescence enhancement. For non-cancerous cells, the telomerase substrate-containing strand is already dissociated with extracellular alkaline pH and cannot enter into cells to achieve subsequent docking event. DTDA well distinguishes cancerous cells from non-cancerous cells in which telomerase are both expressed. The strategy can provide a more reliable way for telomerase-based cancer diagnosis and telomerase oncogenic study. [Display omitted] • A dynamic DNA assembly is designed for responding telomerase in cancerous cells. • The intracellular telomerase catalyzes on the internalized integral assembly with a docking event. • The DNA assembly distinguishes cancerous cells from non-cancerous cells that express telomerase. • The strategy provides a reliable way for telomerase-based cancer diagnosis and telomerase oncogenic study. [ABSTRACT FROM AUTHOR]