Bioluminescence reaction, which uses luciferin, Mg²⁺-ATP and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by the luciferase and emit visible light. One of the most promising applications of firefly luciferase is biosensors. In order to develop an apoptosis biosensor based on caspase 3/7, we have generated 3 forms of circularly permuted variants of Photinus pyralis firefly luciferase and a relatively good tolerance toward disruption of the polypeptide chain by introduction of new termini were found. Two forms of circular permuted luciferases showed significant activity enhancement in comparison with control after exposure to caspase 3. Moreover, the effect of circular permutation and also the length of inserted peptide (caspase 3/7 recognition sites) in structure of firefly luciferase were analyzed using circular dichroism and fluorescence spectroscopy.