Luminescent nanomaterials originate from the convergence of nanomaterials and luminescence. Before the advent of luminescent nanomaterials, biosensing and imaging relied solely on organic dyes, enzyme-based assays, and radioisotopes. However, the photobleaching issues of organic dyes, the instability of enzyme-based assays, and the radiation hazards of radioisotopes necessitated the development of luminescent nanomaterials for enhanced biosensing and imaging. The emergence of luminescent nanomaterials with distinct optical properties is driving progress in biosensing and bioimaging. Nanomaterials, with their unique size-dependent properties, are at the forefront of the swiftly advancing field of nanotechnology, which makes them highly sought after in diverse fields and has led to increased attention on their applications in biosensing and bioimaging. Nanomaterials that can visualize biological processes and interactions at the cellular or subcellular level are highly valued in biomedical applications. A spectrum of luminescent nanomaterials has been developed in recent days, such as quantum dots, noble metal nanoclusters, upconversion nanoparticles, perovskite nanostructures, bioluminescent proteins, and lanthanide-doped materials with excellent sensitivity, which have been tailored for biosensing and bioimaging applications. These luminescent nanoparticles possess distinct absorption and emission characteristics, tunability, high surface area for functionalization, and quantum confinement, which can be tailored for probing biomolecules within cells or tissues. Biosensing has been a fundamental process in the nature for ages, with the nose detecting scents and the tongue identifying tastes. Over time, researchers have developed biosensors that mimic biological recognition using natural or engineered bioreceptors. By combining these elements with various detection methods, biosensors have become powerful tools for highly selective chemical analysis and bioanalysis. Similarly, researchers have developed various tools using luminescent naomaterials that are revolutionizing biomedical imaging. This chapter discusses the process of luminescence and emphasizing the application of luminescence nanoparticles in the advancing fields of biosensing of different analytes and bioimaging.