The thyroid hormones play a crucial role in regulating various physiological processes in the human body. They have a wide range of effects that impact metabolism, growth, development, and overall homeostasis. The current study aimed to investigate the levels of HbA1c and various biomarkers in different patient groups. Two groups of patients were included in the study, each consisting of 30 patients, encompassing both genders. One group comprised patients with type 2 diabetes mellitus (T2DM) who were receiving insulin treatment, while the other group comprised patients with T2DM who were not receiving insulin treatment. These patient groups were compared to a control group of participants from both genders. The research employed the colourimetric method to measure HbA1c levels in all groups. Additionally, they utilized the Enzyme-Linked Immunosorbent Assay (ELISA) method to measure the levels of insulin, and T4 in all groups. The study also involved comparing these biomarkers between groups and examining the effect of insulin levels on thyroid hormones. The mean±SD values were 4.4867±1.02 µg/dl and 3.2367± 0.78 µg/dl for the T2DM groups with and without insulin treatment, respectively, while it was 7.9033±0.29 µg/dl in the control group. These findings provide valuable insights into the relationship between insulin levels and thyroid hormones, shedding light on the complex interplay between these two physiological systems. Overall, the impact of insulin on thyroid hormone regulation underscores the intricate interplay between endocrine systems and highlights the need for a comprehensive understanding of these interactions to optimize patient care and improve health outcomes.