Rare earth doped upconversion nanoluminescent materials (UCNP) can convert low-frequency photons into high-frequency photons, usually near-infrared light excitation and visible light emission. This unique optical property makes it promising for biological applications. In recent years, UCNPs have made significant progress in the imaging and sensing fields. This paper reviews the important progress in synthesis, surface modification, and bioassay using UCNPs, covering important advances in biological detection, and including upconversion fluorescence-based temperature, ions, small molecules, and important proteins and nucleic acids.