Spatial imaging of RNAs in single cells is extremely charming for deciphering of regulatory mechanisms in multiple migration modes during tumor metastasis. Herein, enzyme-free-mediated cascade amplified nanoprobes were designed for in situ single-molecule imaging of dual-microRNAs (miRNAs) in switchable migrating cells. Differential expression and localization of dual-miRNAs were clearly exhibited in multiple cell lines attributed to enhanced sensitivity via the cascade signal amplification strategy. Significantly, in situ three-dimensional (3D) imaging of dual-miRNAs in transition of cell migration phenotypes was successfully reconstructed in both non-confined and confined microenvironments in vitro, of which differential spatial distribution was observed in a single cell. This is very promising for exploring key roles of spatial RNA distribution in migrating cells at the single-molecule level, which will advance revealing the molecular mechanism and physical principle in 3D cell migration in vivo.