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Bioluminescence refers to the emission of light from a living system in which photoproteins such as luciferase enzymes oxidize their substrates to produce light. Because of its high-sensitivity and low-toxicity, bioluminescence imaging (BLI) is particularly useful for in vitro assays and in vivo small animal imaging. It provides a powerful tool to study various important biological questions and processes including gene and protein expression, protein-protein interactions, protein-nucleic acid interactions, and cell signaling pathway functions. This review highlights some of the latest developments in the design and applications of molecular probes for BLI.