Near infrared (NIR) excited lanthanide-doped upconversion nanoparticles (UCNPs) are emerging as a new type of fluorescent tag for biological applications, which can emit multi-photon ultraviolet, visible or NIR luminescence for imaging or activation of photosensitive molecules. Here, we present a comprehensive review on recent advances of UCNPs for a manifold of biological applications, including upconversion mechanisms, building bright multicolor upconversion nanocrystals, single nanoparticle and super resolution imaging, in vivo optical and multimodal imaging, photodynamic therapy, light-controlled drug release, biosensing, and toxicities. Our perspectives on the future development of UCNPs are also described.