Lanthanide-doped nanoparticles exhibit unique optical properties, such as a long luminescence lifetime (up to several milliseconds), sharp emission peaks, and upconversion luminescence over the range of wavelengths from near-infrared to visible. Exploiting these optical properties, lanthanide-doped nanoparticles have been widely utilized for cellular and small animal imaging with the absence of background autofluorescence. In addition, these nanoparticles have advantages of high signal-to-noise ratio for highly sensitive and selective diagnostic detection. In this review, we summarize and discuss recent progress in the development of highly sensitive diagnostic methods using lanthanide-doped nanoparticles. Combined with a smartphone, portable luminescence detecting platforms could be widely applied in point-of-care tests.