Artificial DNA circuits represent a versatile yet promising toolbox for in situ monitoring and concomitant regulation of diverse biological events within live cells. Nonetheless, their performance is significantly impeded by the diffusion‐dominated slow reaction kinetics and the uncontrollable off‐target activation. Herein, a self‐localized cascade (SLC) circuit is reported for the robust and efficient microRNA (miRNA) analysis in living cells. The SLC circuit consists of the cell‐specific localization module and the analyte‐specific signal amplification module. By integrating the reaction probes of these two modules, the complexity of the system is reduced to realize the responsive co‐localization of circuitry probes and the simultaneous cascade signal amplification. Taking advantage of the specifically activated, self‐localized, and cascade design, the SLC circuit successfully achieves the robust miRNA‐21 (miR‐21) imaging and the accurate cells differentiation. Moreover, the reverse regulation mechanism is successfully explored between messenger RNA (mRNA) and miRNA through the engineered SLC circuit and further elucidates the underlying signaling pathways between them. Therefore, the SLC circuit provides a powerful tool for the sensitive detection of intracellular biomolecules and the study of the corresponding cell regulatory mechanisms.