Bioluminescence (BL) is an excellent optical readout platform that has great potential to be utilized in various bioassays and molecular imaging. The advantages of BL-based bioassays include the long dynamic range, minimal background, high signal-to-noise ratios, biocompatibility for use in cell-based assays, no need of external light source for excitation, simplicity in the measurement system, and versatility in the assay design. The recent intensive research in BL has greatly diversified the available luciferase-luciferin systems in the bioassay toolbox. However, the wide variety does not promise their successful utilization in various bioassays as new tools. This is mainly due to complexity and confusion with the diversity, and the unavailability of defined standards. This review is intended to provide an overview of recent basic developments and applications in BL studies, and showcases the bioanalytical utilities. We hope that this review can be used as an instant reference on BL and provides useful guidance for readers in narrowing down their potential options in their own assay designs.