Chiral self-assembled nanomaterials with biological applications have attracted great interest. In this study, DNA-driven gold-upconversion nanoparticle (Au-UCNP) pyramids were fabricated to detect intracellular microRNA (miRNA) in real time. The Au-UCNP pyramids are doubly optically active, displaying strong plasmonic circular dichroism (CD) at 521 nm and significant luminescence in 500–600 nm, and therefore can be monitored by both of them. CD will decrease while the luminescence intensity increases in the presence of miRNA. The experimental results show that the CD intensity had an outstanding linear range from 0.073 to 43.65 fmol/10 μgRNA and a limit of detection (LOD) of 0.03 fmol/10 μgRNA, whereas the luminescence intensity ranged from 0.16 to 43.65 fmol/10 μgRNA with a LOD of 0.12 fmol/10 μgRNA. These data indicate that the CD signal is much more sensitive to the concentration of miRNA than the luminescent signal, which is attributed to the strong CD intensity arising from the spin angular momentum of the photon interaction with chiral nanostructures and the plasmonic enhancement of the intrinsic chirality of DNA molecules in the pyramids. This approach opens up a new avenue to the ultrasensitive detection and quantification of miRNA in living cells.