Objective: The present research was conducted to evaluate the negative effects of nicotine powder on the blood physiology, and biochemical and histological alterations of Labeo rohita. Materials and Methods: Fish were divided into four groups (1–4). Fish groups 2, 3, and 4 were exposed to different concentrations of nicotine, such as 0.75, 1.25, and 1.75 mg/l, while group 1 acted as a control. To find out the long-term impact of nicotine on body physiology, we conducted a 42-day experiment. After the completion of the experiment, hematology, biochemical assays, and histology were done. Results: Results revealed a considerable increase in HGB, red blood cells, WBCs, hematocrit, mean corpuscular volume, red cell distribution width -SD, procalcitonin, neutrophils, lymphocytes, monocytes, triglycerides, total cholesterol, low-density lipoprotein, very low-density lipoprotein, alanine transaminase, aspartate aminotransferase, globulin, thyroid stimulating hormone, BUN, creatinine, and blood glucose levels, whereas mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, RDW, platelet, high-density lipoprotein, albumin, total proteins, and T3 levels were significantly (p ≤ 0.05) decreased in exposed fish as compared to control group fish. Histological alterations showed that exposure to smokeless nicotine causes deleterious and degenerative effects in the liver, kidney, and gills of exposed fish. Conclusion: Nicotine administration in fish results in adverse effects on different biochemical and hematological parameters and causes histological alterations in some vital organs of exposed fish. [J Adv Vet Anim Res 2024; 11(2.000): 463-473]