Terbutryn was discovered to cause cytogenetic and DNA damage in newly isolated human peripheral blood leukocytes in vitro. Terbutryn is a triazine herbicide that has exceptional herbicidal action. The precise mechanisms of action of Terbutryn are unknown. Serum proteins alter the bio-distribution of several endogenous and exogenous substances. Because of all of these potentially hazardous effects, a responsive and cost-effective approach is required to comprehend the in vitro investigation of the interaction between Terbutryn herbicide and bovine serum albumin for understanding their possible molecular consequences. Here, in this work interaction of Terbutryn with bovine serum albumin were studied via a series of spectroscopic methods such as Ultraviolet spectroscopy (UV), steady-state fluorescence spectroscopy, and Molecular docking techniques. The experimental results of the fluorescence quenching mechanism of BSA with Terbutryn was a static quenching. The Stern-Volmer constant (Ksv) of Terbutryn with BSA was 1.64×103 dm3 mol−1 at 298 K. Based on fluorescence quenching measurements, the site binding constants (Kb = 4.88×101 dm3 mol−1) and number of binding sites (n~1) were calculated at 298 K. The competitive experiment results of molecular docking studies confirmed that binding of Terbutryn with BSA at site I (subdomain IIIA). All of these findings suggested that Terbutryn can successfully bind to BSA and be carried throughout the body and removed. This study presented a plausible model that helped us better comprehend the transportation, distribution, and hazardous effects of Terbutryn as it diffused into blood serum. It may serve as a valuable benchmark for future new drug development.