Cancer cells utilize a range of migration modes to navigate through a confined tissue microenvironment in vivo, while regulatory roles of key microRNAs (miRNAs) remain unclear. Precisely engineered microconfinement and the high spatial-resolution imaging strategy offer a promising avenue for deciphering the molecular mechanisms that drive cell migration. Here, enzyme-free signal-amplification nanoprobes as an effective tool are developed for three-dimensional (3D) high-resolution profiling of key miRNA molecules in single migrating cells, where distinct migration modes are precisely driven by microconfinement-engineered microchips. The constructed nanoprobes exhibit intuitive and ultrasensitive miRNA characterization in vitro by virtue of a single-molecule imaging microscope, and the differential expression and intracellular locations in different cell lines are successfully monitored. Furthermore, 3D spatial distribution of miR-141 at high resolution in flexible phenotypes of migrating cells is reconstructed in the engineered biomimetic microenvironment. The results indicate that miR-141 may be involved in the metastatic transition from a slow to a fast migration state. This work offers a new opportunity for investigating regulatory mechanisms of intracellular key biomolecules during cell migration in biomimetic microenvironments, which may advance in-depth understanding of cancer metastasis in vivo.