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A biosensor that can detect biomarkers accurately, quickly, and conveniently is important for the diagnosis of various diseases. However, most of the existing detection methods require sample extraction, which makes it difficult to detect and image intracellular molecules or to detect two different types of biomarkers simultaneously. In this study, we constructed a DNA tetrahedral nanoprobe (DTP) capable of detecting both miR378 and telomerase, both of which are tumor markers. In the presence of miR378, FAM on the molecular beacon of DTP fluoresced via Förster resonance energy transfer (FRET), and the limit of detection was 476 pM with excellent specificity. When present, telomerase binds to telomerase substrate (TS) primers, extending the repeat sequence (TTAGGG)n to trigger Cy3 fluorescence. A strong linear relationship existed between the fluorescence intensity of Cy3 and the number of HeLa cells. The limit of detection was 800 HeLa cells. In addition, DTP was less cytotoxic to and biocompatible with HeLa cells and fluoresced only in cancer cells, which can help to sensitively distinguish between normal and cancer cells. In conclusion, DTP can simultaneously detect the content of miR378 and activity of telomerase and realize intracellular imaging, which has broad application prospects in early cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]