According to the NIH roadmap, optical molecular imaging has an instrumental role in the development of molecular medicine. Great efforts, including those with bioluminescent imaging techniques, have been made to understand the linkage between genes and phenotypic expressions in normal and disease biology. Currently, bioluminescent techniques are widely used in small animal studies. However, most of the current bioluminescent imaging techniques are done in the 2D mode. In this overview, we review bioluminescence tomography (3D mode), elaborate on its principle and multi-spectral extension, describe associated image unmixing and normalization techniques, and discuss a number of directions for technical improvements and biomedical applications.