Lanthanides are particularly effective in their clinical applications in magnetic resonance imaging and diagnostic assays. They have open-shell 4f electrons that give rise to characteristic narrow, line-like emission which is unique from other fluorescent probes in biological systems. Lanthanide luminescence signal offers selection of detection pathways based on the choice of the ion from the visible to the near-infrared with long luminescence lifetimes that lend themselves to time-resolved measurements for optical multiplexing detection schemes and novel bioimaging applications. The delivery of lanthanide agents in cells allows localized bioresponsive activity for novel therapies. Detection in the near-infrared region of the spectrum coupled with technological advances in microscopies opens new avenues for deep-tissue imaging and surgical interventions. This review focuses on the different ways in which lanthanide luminescence can be exploited in nucleic acid and enzyme detection, anion recognition, cellular imaging, tissue imaging, and photoinduced therapeutic applications. We have focused on the hierarchy of designs that include luminescent lanthanides as probes in biology considering coordination complexes, multimetallic lanthanide systems to metal–organic frameworks and nanoparticles highlighting the different strategies in downshifting, and upconversion revealing some of the opportunities and challenges that offer potential for further development in the field.