Display omitted •The use of a DNA nanotetrahedron-assisted aptasensor greatly enhances the specificity of target detection.•The use of quasi-ZIF-67 greatly improved the MB loading efficiency for enhancing the detection sensitivity.•The aptasensor exhibited high detection sensitivity with a low detection limit (3 pg/mL) for EpCAM.•This electrochemical aptasensor offers a cost-effective alternative to flow cytometry. The cell surface protein epithelial cell adhesion molecule (EpCAM) has oncogenic effects, and its overexpression is strongly correlated with tumor development. Here, an electrochemical method is proposed to detect and identify EpCAM at the level of purified proteins and living cells. The core concept of this method is the use of DNA nanotetrahedron (NTH)-based capture probes and aptamer-modified quasi-ZIF-67@Au@methylene blue (QZIF-67@Au@MB@APT) as signal probes. In our design, NTH-assisted targeted immobilization can greatly improve the accessibility of nucleotide-containing artificial aptamers to suspended targets and the specificity of the target EpCAM. Moreover, following partial deligandization, ZIF-67 was converted to QZIF-67 with an extensively improved porous structure that significantly enhanced the loading efficiency of signaling molecules and thus led to a remarkable increase in detection sensitivity. QZIF-67@Au@MB can be integrated with an aptamer to act as a signal reporter with an electrochemical sensing platform for EpCAM detection. By tracing the electrochemical signals from the QZIF-67@Au@MB@APT, the method demonstrated the detection of targets as low as 3 pg/mL within a wide linear range from 0.005 to 100 ng/mL. We have successfully applied this aptasensor for in situ characterization of EpCAM on the cellular surface and monitoring alterations in EpCAM expression during drug treatment, which provides a low-cost but robust alternative to highly expensive and experience-dependent flow cytometry. Overall, electrochemical characterization may provide promising technical support for the precise management of tumors.