A New photoprobe based on tris(3-acetylindole) terbium (III) complex (TbAcI) doped in polyethylene glycol (PEG) thin-film was synthesized, and employed as a novel, simple, sensitive, accurate, and precise method for the determination of Epinephrine (EPI) in different serum samples. The results obtained from the Stern-Volmer bimolecular quenching analysis was attributed to the presence of EPI in the surrounding of the emissive TbAcI. The calibration plot was prepared for the concentration range of 1-1500 pg/mL of EPI with a correlation coefficient of 0.99 and a detection limit of 0.1 pg/mL. That may aid in assessing the activity of the adrenal gland and then the diagnosis of optimal diseases that may be raised due to the abnormal ranges in human blood.