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New tools for single‐cell interrogation enable deeper understanding of cellular heterogeneity and associated cellular behaviors and functions. Information of RNA expression in single cell could contribute to our knowledge of the genetic regulatory circuits and molecular mechanism of disease development. Although significant progresses have been made for intracellular RNA analysis, existing methods have a trade‐off between operational complexity and practical feasibility. We address this challenge by combining the ionic current rectification property of nanopipette reactor with duplex‐specific nuclease‐assisted hybridization chain reaction for signal amplification to realize a simple and practical intracellular nanosensor with minimal invasiveness, which enables single‐cell collection and electrochemical detection of intracellular RNA with cell‐context preservation. Systematic studies on differentiation of oncogenic miR‐10b expression levels in non‐malignant breast cells, metastatic breast cancer cells as well as non‐metastatic breast cancer cells were then realized by this nanotool accompanied by assessment of different drugs effects. This work has unveiled the ability of electrochemistry to probe intracellular RNA and opened new opportunities to study the gene expression and heterogeneous complexity under physiological conditions down to single‐cell level. [ABSTRACT FROM AUTHOR]