A unique bioluminescent imaging probe is introduced for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. A full-length luciferase is sandwiched between two proteins of interest via minimal flexible linkers. The ligand-activated PPIs append intramolecular tension to the sandwiched luciferase, boosting or dropping the enzymatic activity in a quantitative manner. This method guides construction of a new lineage of bioassays for determining molecular tension appended by ligand-activated PPIs. The summary of the method is: •Molecular tension appended by protein-protein interactions (PPI) is visualized with a luciferase.•Estrogen activities are quantitatively illuminated with the molecular tension probes.•Full-length Renilla luciferase enhances the optical intensities after bending by PPI.