Labeo rohita were exposed to amoxicillin at a concentration of 1 mg/L (Treatment -I) and 0.5 mg/L (Treatment-II) for a period of 35 days. Numerous alterations were found in amoxicillin treatment groups when compared to the control group. Hemoglobin (Hb), hematocrit (Hct), and erythrocytes (RBCs) levels were significantly ( P < 0.05) decreased. Leukocytes (WBC), mean cell volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) levels were significantly ( P < 0.05) increased. In the plasma and gill tissues, ionic compounds (sodium, potassium, and chloride) levels were significantly declined throughout the treatment period. The plasma biochemical profiles were significantly altered: glucose level remained low (except at the end of 7 th day in Treatment -I) till 35 days of the treatment period. Biphasic trend occurred in the protein level, significant increase was observed on 7 th and 28 th day (Treatment -I and -II), and 35 th day (Treatment -I), and in remaining days its level was found to be decreased. Glutamate oxaloacetate transaminase (GOT) activity in the plasma was inhibited significantly, whereas in the gill, liver, and kidney tissues the enzyme activity was elevated. Plasma glutamate pyruvate transaminase (GPT) activity was inhibited throughout the study period. GPT activity in the gill was found to be elevated during the treatment period. Liver GPT activity was elevated in all the treatments except 28 th (Treatment-I) and 35 th day (Treatment-I, and II). GPT activity in the kidney was elevated (except 14 th day in Treatment-II). Lactate dehydrogenase (LDH) activity was inhibited in plasma (except 14 th day in Treatment-II), gill, liver (except 7 th day in Treatment-I), and kidney tissues significantly ( P < 0.05). The present study emphasizes that amoxicillin at 1 and 0.5 mg/L concentrations affects the hematological/biochemical/electrolytes/enzymological parameters of fish and these biomarkers serve as an effective test system for environmental risk assessment of pharmaceuticals in the aquatic environment.