As a means for visualizing molecular physiology within living cells, new strategies are emerging for engineering luciferases into intracellular biosensors. These biosensors can be classified as bimolecular, relying on complementation of luciferase fragments, or intramolecular, relying on domain insertion within the luciferase structure. Multiple design strategies have recently surfaced for the development of intramolecular sensors, allowing dynamic detection of small molecules or post-translational modifications within cells. Building on successes achieved in cell culture, these sensors are now beginning to reveal molecular processes within living organisms.