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Rare earth ion– (RE) and transition metal (TM) ion–doped inorganic luminescent nanocrystals (NCs) have recently evoked considerable interest owing to their superior optical features such as long-lived downshifting luminescence, near-infrared–triggered anti-Stokes upconverting luminescence, and excitation-free persistent luminescence, which make them extremely suitable for use as sensitive luminescent nanoprobes for background-free biosensing. A fundamental understanding of the photophysics of RE- and TM-doped luminescent NCs is of vital importance for optimizing their optical performance and discovering novel optical properties. In this review, we focus on our recent efforts on the development of RE- and TM-doped luminescent NCs from their fundamental physicochemical properties to biodetection, including the controlled synthesis, electronic structure, optical property designing, and their promising application for in vitro luminescent biodetection of various disease markers. Future opportunities and challenges toward this intriguing field are also envisioned.