Bioluminescence imaging (BLI), an optical preclinical imaging modality, is an invaluable imaging modality due to its low-cost, high throughput, fast acquisition times, and functional imaging capabilities. BLI is being extensively used in the field of cancer imaging, especially with the recent developments in genetic-engineering, stem cell, and gene therapy treatments. The purpose of this paper is to provide a comprehensive review of the principles, developments, and current status of BLI in cancer research. This paper covers the fundamental BLI concepts including BLI reporters and enzyme-substrate systems, data acquisition, and image characteristics. It reviews the studies discussing the use of BLI in cancer research such as imaging tumor-characteristic phenomena including tumorigenesis, metastasis, cancer metabolism, apoptosis, hypoxia, and angiogenesis, and response to cancer therapy treatments including chemotherapy, radiotherapy, immunotherapy, gene therapy, and stem cell therapy. The key advantages and disadvantages of BLI compared to other common imaging modalities are also discussed.