Background: Assessment of changes in structure and activity of catalase, as an antioxidant enzymatic protein, in combination with glucose and essential unsaturated fatty acids is the aim of present study. Materials and Methods: In order to investigate the activity and structure of catalase, a solution of this enzyme with 10 mg/mL concentration in phosphate buffer (0.1 M) and pH = 7.4, in the presence and absence of 50 mmol glucose was prepared, filtered, and then incubated for 4 months at 37°C with and without fatty acids including linolenic, linoleic, and arachidonic acids, separately. Samples were taken from each tube of solution, every 14 days for 4 months, to assess the fluorescence emission, circular dichroism (CD) and activity. Results: According to the results, catalase showed increase in fluorescence emission and decrease in activity after incubation with glucose in comparison with pure protein. Moreover, catalase showed alteration in CD after incubation with glucose during 4 months. After incubation with glucose and each of the mentioned unsaturated fatty acids, alterations of catalase were neared toward normal level in fluorescence emission, CD, and activity in comparison with pure protein. Conclusion: Functional and structural protection of catalase against damages from hyperglycemic environment with addition of essential unsaturated fatty acids was proved in our investigation.