An upconversion nanoparticle (UCNP)-based fluorescence resonance energy-transfer (FRET) strategy is normally restricted by the complicated preparations, low energy-transfer efficiency, and the challenge on improving specificity. Herein, simple DNA-functionalized UCNPs were designed as energy donors for constructing a FRET-based probe to detect the liver-specific microRNA 122 (miR-122). To improve FRET efficiency, UCNPs were constructed with confined core-shell structures, in which emitting ions were precisely located in the thin shell to make them close enough to external energy acceptors. Subsequently, capture DNA was simply functionalized on the outer surface of UCNPs based on ligand exchange that contributed to shortening the energy-transfer distance without extra modification. To gain high specificity, the donor-to-acceptor distance of FRET was controlled by a sandwich DNA hybridization structure using two shorter DNAs with designed complementary sequences (capture DNA and dye-labeled report DNA) to capture the longer target of miR-122. Therefore, the sensitive detection of miR-122 was achieved based on the decreased signals of UCNPs and the increased signals of the dye labeled on reported DNA. With good biocompatibility, this method has been further applied to cancer cell imaging and in vivo imaging, which opened up a new avenue to the sensitive detection and imaging of microRNA in biological systems.