Some unique living organisms produce visible light from chemical energy in a process called bioluminescence. Bioluminescent organisms express luciferase enzymes which catalyze chemical reactions in which luciferases convert their substrates and produce visible light. Different bioluminescent organisms contain different luciferase enzyme/substrate systems. For creating an assay system, genes encoding selected luciferase reporter and any protein, can be fused via cDNA synthesis and then luciferase-fused protein can be traced in living organism or in cell culture, depending on bioluminescence glowing (radiation). Besides, in a predetermined setup, progression phases of experimentally created infections of any bacteria, virus, parasite or fungus species which transfected with luciferase encoding gene can easily be traced via bioluminescence. Any bioluminescence assay system is composed from three elements: transfer of luciferase gene and injection of its substrate material to animal subject, and acquiring/processing light signals by charge coupled device (CCD) camera. In bioluminescence resonance energy transfer (BRET) system which is particularly used in protein-protein interaction (PPI) studies, closely positioned or interacting labeled-proteins give both bioluminescent and fluorescent signals. In comparison to other protein-assay techniques, bioluminescence imaging is simple, non-invasive, cost-effective and convenient technique which is promising in terms of finding more usage areas in the future. In this paper, besides a review of important studies focused on the subject, general knowledge about basic principles of bioluminescence, various bioluminescence-creating enzyme-substrate systems and bioluminescence imaging (BLI) modalities were provided.