Present study was designed to evaluate in vitro antioxidant activity in heart and serum Glucose, HbA1c of Hesperidin on isoproterenol induced myocardial infarction in normal and diabetic in rats. Hesperidin (100 mg/kg, p.o) was administered for 28 days in rats injected with single dose of Streptozotocin (65 mg/kg, i.p, STZ) and nicotinamide (110 mg/kg, i.p, NIC) and after isoproterenol (200mg/kg, s.c., ISO) induced myocardial infarction in diabetic rats on 29th and 30th day. At the end of experimental period (i.e. on the day 31) heart tissue sample of each rat was collected and antioxidative parameter carried out for further estimations. Administration of STZ–NIC in rats showed a significant (P<0.001) increased in the levels of serum glucose, glycosylated heamoglobin (HbA1c). At the end of experimental period the level of malondialdehyde formation/ lipid peroxidation (LPO) and nitrite level in heart tissue was significantly increased. Whereas, the activity of biomarkers of oxidative stress such as reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) were found to be decreased significantly compared to control rats. Treatment with Hesperidin significantly restored GSH level, SOD as well as catalase activity and reduced lipid peroxidation and nitrite in compared to diabetic control group. This study concluded that HES at 10 mg/kg may show reduced oxidative stress in heart on isoproterenol induced myocardial infarction in type 2 diabetic rats.