Trivalent lanthanide ions (Ln(3+)) doped inorganic nanocrystals (NCs) have currently attracted reviving interest and come to the forefront in nanophotonics owing to their potential applications in diverse fields such as luminescent biodetection and bioimaging. As an alternative to conventional biolabels, Ln(3+)-doped NCs show superior features including large stokes shift, multicolor fine-tuning, narrow emission band widths, high photostability, and low toxicity. Particularly, the long-lived luminescence and distinct upconversion (UC) of Ln(3+)-doped NCs are desirable for various bioapplications. The long-lived luminescence of Ln(3+) combined with time-resolved technique can efficiently suppress the interference from short-lived background, resulting in a high signal-to-noise ratio (S/N) and background-free measurements. Near-infrared excited UC emissions of Ln(3+) can bring no autofluorescence and no photodamage to cells or tissues, and thus UC NCs have been regarded as one of the most useful in vivo optical contrast agents. In this review, we outline the most recent development of Ln(3+)-doped NCs as biolabels from the controlled synthesis and surface functionalization of NCs to their bioapplications in heterogeneous and homogeneous biodetection as well as in vitro and in vivo bioimaging.