Simultaneous detection of multiple types of microRNA (miRNA) is of great significance in early cancer diagnosis. In this study, rare-earth-doped CaF2 upconversion nanocrystals (CaF2: RE3+, namely Ho3+ or Tm3+ and Yb3+) are formed within the mesopores of silica nanoparticles (CaF2: RE3+@MSN), in an in-situ manner, by capillary effect and thermo-decomposition method. Comparing to current upconversion nanoparticles, the amorphous and mesoporous nature of CaF2: RE3+@MSN provides abundant active groups and high specific surface area for feasibly immobilizing oligonucleotide probe. The simultaneous and selective detection of multiple miRNAs was achieved based on the photoluminescence spectra of different CaF2: RE3+@MSN conjugated with DNA probes in the same solution. The results indicated that the proposed approach holds significant potential for multiple miRNA detection and offers an opportunity for cancer early diagnosis.