Objectives <p>This study focused on determining the new marker Klotho and its correlation with other parameters in thyroid dysfunction.</p> Methods <p>Seventy-seven thyroid dysfunction patients were recruited during the period (from Aug 2024 to Dec 2024) with an age range (33–56) years. The patient group was subdivided into two groups: group I (21) Patients with Hyperthyroidism and group II (56) Patients with Hypothyroidism, for comparison, 30 healthy individuals were included as a control. The protein level Klotho was estimated using ELISA. Biochemical parameters; blood urea, creatinine, and lipid profile were measured spectrophotometrically. T3, T4 and TSH were determined using ELFA kit.</p> Results <p>Serum levels of Klotho protein were significantly lower (<i>p</i>-value &lt; 0.001) in group I compared to the control group. There was a significant difference (<i>p</i> &lt; 0.001) in the Klotho protein levels in group II compared with the control group. Klotho protein was negatively correlated to TSH, creatinine concentrations, and age. The AUC was excellent with high diagnostic accuracy in differentiating newly diagnosed thyroid dysfunction from the healthy subject group.</p> Conclusions <p>Serum Klotho levels in patients with thyroid dysfunction significantly decreased, particularly in hypothyroidism patients. These levels are inversely correlated with thyroid-stimulating hormone levels and age.</p>