Persistent luminescence phosphors are a novelgroup of promising luminescent materials with afterglow prop-erties after the stoppage of excitation. In the past decade, persis-tent luminescence nanoparticles (PLNPs) with intriguing optical properties have attracted a wide range of attention in various areas. Especially in recent years, the development and applica-tions in biomedical fields have been widely explored. Owing to the effcient elimination of the autofluorescence interfer-ences from biotissues and the ultra-long near-infrared afterglow emission, many researches have focused on the manipulation of PLNPs in biosensing, cell tracking, bioimaging and cancer therapy. These achievements stimulated the growing interest in designing new types of PLNPs with desired superior charac-teristics and multiple functions. In this review, we summarize the works on synthesis methods, bioapplications, biomembrane modification and biosafety of PLNPs and highlight the recent advances in biosensing, imaging and imaging-guided therapy.We further discuss the new types of PLNPs as a newly emerged class of functional biomaterials for multiple applications. Finally, the remaining problems and challenges are discussed with suggestions and prospects for potential future directions in the biomedical applications.