<p>Indiscriminate use of pesticides not only pollutes the environment but also produces detrimental impacts on non-target organisms. Emamectin benzoate (EMB) is an important pesticide used for pest control in both agriculture and veterinary medicine. However, aquatic animals are endangered by the continuous influx of EMB into water bodies. The present study examined the long-term impacts of EMB on the haemato-immuno-antioxidant biomarker pattern, histopathological alteration, and amino acid-fatty acid profiling of <i>L. rohita</i> exposed to EMB; 0 (control), 1.82 µg L⁻¹ (EMB1), and 9.1 µg L⁻¹ (EMB2) for 30 days. This is the first-ever study investigating amino acid and fatty acid profiles and histopathological alterations in <i>L. rohita</i> subjected to EMB. The results showed that EMB exposure significantly altered biomarker levels in a dose-dependent manner. The stress biomarkers levels (SOD, catalase, GST, AST, ALT, triglyceride, glucose, and cortisol) were increased, but the immune responses like serum albumin, globulin, IgM, C3, and C4 were reduced. Histopathological observation showed that EMB incited liver, gill and kidney damage. The reduction of most amino acids is probably due to the synthesis pathway inhibition of amino acids in EMB-exposed fish. The reduced ∑PUFA proportion in treated fish was due to the susceptibility of C20:5 and C22:6 to peroxidation by EMB. In the context of ecotoxicology risk assessment, the alteration in multiple biomarkers is an adaptive response of fish exposed to EMB and that could impose risks to the natural population subjected to food quality and safety.</p>