The pathology of many diseases arises from oxidative stress and cell destruction. Antioxidant application is one of the most important ways for oxidative stress prevention in the cells and its consequent effects. The present study investigated the natural antioxidants inhibitory effects of thymol and carvacrol on human hemoglobin treated with tartrazine. Purified hemoglobin from human blood samples was treated with tartrazine alone or in combination with mentioned natural antioxidants (thymol and carvacrol). Treated samples were picked up at regular time intervals and changes were followed by UV–visible and fluorescence spectroscopic assays, and circular dichroism spectroscopy (CD). The result of fluorescence spectroscopy revealed that thymol and carvacrol prevented the production of heme-degradation products and advanced glycation end products (AGEs) caused by hemoglobin oxidation with tartrazine. The results of UV–visible and fluorescence spectroscopy revealed the positive effect of these antioxidants on preserving Hb folding, heme, and especially the porphyrin ring surrounding the microenvironment. The results of the circular dichroism (CD) assay showed the protection of alpha helix structure in hemoglobin treated with thymol and carvacrol compared to the control sample. The mentioned antioxidants caused hemoglobin resistance against tartrazine's destructive effect by preventing both heme degradation and glycemic toxins formation and thus reducing the rate of oxidative processes. This matter can be important for various pharmaceutical, health, and cosmetic industries.