As a reliable, sensitive, convenient and non-invasive in vivo imaging technology, bioluminescence imaging (BLI) has been extensively applied in detecting physiological and pathological processes in biomedical research, including pathogen detection, tumor growth and responses to therapy patterns of gene regulation, measurements of protein–protein interactions and ADMET (absorption, distribution, metabolism, excretion and toxicity). Among various bioluminescence systems, firefly luciferase-luciferin is powerful and popular to noninvasively visualize molecular and cellular features in living mice. Some drawbacks of natural substrates D-luciferin and aminoluciferin, however, hamper the application of bioluminescent imaging with firefly luciferase, such as short red-light (tissue-penetrating) emission, short in vivo bioluminescence time and low blood-brain barrier permeability.