<p>Luciferase complementation assays have emerged in 2001 as a useful tool to analyze biological processes through diverse biological assays such as cellular studies and in vivo imaging. The assay has an advantage of wide dynamic ranges, high signal-to-noise ratios, and capability for real-time monitoring of dynamic biological events with a readout of bioluminescence. While it was initially harnessed for detecting protein–protein interactions, biosensors based on luciferase-fragment complementation have achieved significant advancements in their designs, expanding versatility and applicability beyond the initial scope. This review aims to provide a comprehensive overview of designing strategies employed in split luciferase complementation assays and to highlight their diverse bioanalytical applications. Because simple bi-molecular detection of protein–protein interactions by this approach is well-established, this review will focus on introducing diverse sensor designs using the concept of split luciferase complementation.</p> Graphical abstract <p></p>