Butachlor herbicide belongs to the family of chloroacetanilide group, widely used for control of grass and broadleaf weeds in paddy fields however, its repeated application may result in aquatic pollution. Butachlor residue has been detected in aquatic environments, which may produce toxic effects on non-target organisms including fish. Keeping this in mind, the present study was designed to estimate the LC of butachlor (Shaktiman®), and to evaluate the sub-lethal toxicity at two concentrations (12.42 μg L and 62.10 μg L) in Labeo rohita for a period of 24, 48, and 72 h. Fish exposed to butachlor reduced the counts of red blood cells (RBC), haemoglobin (HGB), hematocrit (HCT), and white blood cells (WBC). A significant (p < 0.05) increase in the antioxidant enzyme (superoxide dismutase-SOD, glutathione-s-transferase-GST), and hepatic enzyme (glutamate-oxaloacetate transaminase-GOT, glutamate-pyruvate transaminase-GPT) were noticed in butachlor exposed fish. Heat shock protein 70 (HSP70) and HSP90 in gill; cortisol, protein, albumin, globulin, and triglyceride in serum were increased upon exposure of butachlor. On the contrary, complement 3 (C3) and immunoglobulin (IgM) in serum was found to be decreased compared to control fish. The findings thus suggest that the fish upon exposure to butachlor disrupts the biomarkers which ultimately leads to growth retardation in fish.