Study of 42 days was planned to evaluate the effect of melatonin on biochemical’s parameters in buffalo calves under summer stress. Murrah buffalo bull calves divided into two groups viz., control (CG) and treatment (TG) group (n=6). TG received melatonin @ 18 mg/50 kg body weight, subcutaneously (s/c), on 1st and 20th day of experiment. Blood samples collected at 7 days interval on 0th, 7th, 14th, 21st, 28th, 35th and 42nd days. The study revealed that in CG animals serum LPO activity at 21st, 28th, 35th and 42nd days, ALT activity at 0th, 14th, 21st, 28th, 35th and 42nd days and AST activity at 35th and 42nd days was found significantly (p<0.05) higher whereas GPx activity at 21st, 28th and 42nd days, GST activity at 21st, 28th, 35th and 42nd days and K level at 28th and 42nd days significantly (p<0.05) lower than TG. Na, Cl, creatinine and urea level did not differed significantly between both groups. It could be concluded from the result of this study that melatonin ameliorate the heat stress in Murrah buffalo calves during summer by affecting the biochemical’s parameters.