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Abstract Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems. Graphical abstract A new caspase-9 biosensor, which has been built by inactivating luciferase through circular permutation and connecting the fragments through a protease site (LEHD). In the presence of active form of caspase-9, peptide backbone cleavage occurs due to the protease activity of caspase-9, thereafter, the spliced luciferase will be active and regain its bioluminescent activity in the presence of substrate and it can be detected by measuring light emission at 482 nm. Therefore, emitted light can be attributed to the caspase-9 activity and apoptosis will be recognized. The biosensor has been constructed for cell-free and cell-based systems. Image 1 Highlights • A luciferase based biosensor for measuring caspase-9 activity has been constructed using circular permutation strategy. • The designed biosensor detects the caspase-9 activity in a cell-free coupled assay system. • The designed biosensor can be implemented as a cell-based biosensor to detect cellular caspase-9 activity and apoptosis. [ABSTRACT FROM AUTHOR]