Pyridine is a basic heterocyclic organic compound. The pyridine ring is present in many important compounds, including agricultural chemicals, medicines and vitamins. Due to their widespread use, bioaccumulation and non-target toxic effects are considered a great risk to human health and the environment. In this study, effects of concentrations (0 (control), 1, 1.5, and 2 g/L) of the ketone “3-Benzoylpyridine” to rainbow trout (Oncorhynchus mykiss) after the determination of LC50-96h value (5.2 g/L) were evaluated. Changes in biomarkers of oxidative DNA damage [8-hydroxy-2′-deoxyguanosine (8-OHdG), apoptosis (Caspase-3)], malondialdehyde (MDA) level, antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), myeloperoxidase (MPO), paraoxonase (PON), and arylesterase (AR)] assessed in brain, liver, gill and blood. Acetylcholinesterase (AChE) activity was performed only in brain tissue. In addition, we performed the micronucleus (MN) test and hematological index [total erythrocyte count (RBC), total leukocyte count (WBC), hemoglobin (Hb), hematocrit (Hct), total platelet count (PLT), mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), and mean cell volume (MCV)] in blood. A significant decrease in brain AChE activity was found with time and dose. While SOD, CAT, GPx, PON, and AR levels decreased but MDA, MPO, 8-OHdG and Caspase-3 levels increased in all tissues (p<0.05). The increase and decrease in hematology parameters (WBC, RBC, Hb, PLT, Hct, MCV, MCH, MCHC) are statistically significant (p<0.05). The results of 3-Benzoylpyridine exposure on trout was obtained for the first time in this research. Overall, our study demonstrated that the differences in sensitivity to acute 3-Benzoylpyridine exposure rainbow trout can be explained on the basis of differential effects of 3-Benzoylpyridine on biochemical changes.