Finely tunable detection of microRNAs (miRNAs) is crucial for personalized medicine and cancer diagnosis, and it is a challenge to construct a tunable sensor for miRNA detection due to the diversity and complexity of cancer patient samples. This study introduces a three-dimensional (3D) DNA walker mediated by a remote toehold strand displacement reaction, which addresses the challenge of achieving tunable detection limits and sensitivities for microRNA-21 (miR-21) at a kinetic level, enabling specific recognition of cancer cells. By engineering a spacer domain within the DNA walker, the kinetics of the strand displacement reaction can be precisely modulated, thereby controlling the walking efficiency and achieving tunable detection with a detection limit range from 32 aM to 290 pM and a broad dynamic range from ∼1100-fold to ∼283,000-fold. This approach leverages the programmability of DNA molecules to design probe structures tailored to experimental requirements, offering high sensitivity and specificity and a wide detection limit. The 3D DNA walker, driven by Zn2+, allows for rapid and accurate imaging of miR-21 in cancer cells, thereby effectively differentiating cancerous cells from normal cells. The proposed method not only demonstrates a high degree of specificity in recognizing miR-21 but also provides a robust foundation for clinical diagnosis and holds promise for applications in personalized medicine, early disease detection, and drug development. These findings underscore the potential of this remote toehold-mediated DNA walker as a powerful tool for real-time monitoring and dynamic detection of disease biomarkers, enhancing the precision and efficacy of therapeutic interventions.