Accurate quantitative, in situ and temporal tracking imaging of tumor-associated miRNAs in living cells could provide a basis for cancer diagnosis and prognosis. In this strategy, a surface-enhanced Raman scattering (SERS)-fluorescence (FL) dual-spectral sensor (DSS) was constructed based on the nanoscale photophysical properties of AuNPs, mediated by functionalized DNA, to achieve rapid imaging of FL and accurate SERS quantification of intracellular miRNAs. The dual-spectrum sensor in the strategy is highly sensitive, specific and reproducibly stable. The LOD values of the dual spectra were 3.58 pM (SERS) as well as 11.8 pM (FL) with RSD values less than 2.69%. The bispectral sensor self-assembled into a trimer by the lapidation of Y-type DNA under the excitation of the target, generating a stable enhanced electric field coupling; and selected adenine located in the enhanced electric field as the reporter molecule, simplifying the labeling process and variables of the Raman reporter molecule, distinguishing it from other traditional methods. This strategy successfully achieved accurate tracking and quantification of miR-21 in cancer cells and showed good stability in the cells. The reported probes are potential tools for reliable monitoring of biomolecular dynamics in living cells.