Hepcidin (HEPC) hormone production is expected to be elevated in cases accompanying iron overload, but the opposite impact of ineffective erythropoiesis in β-thalassemia major (β-TM) patients overrides this effect. The role of the HEPC-to-ferritin (FER) ratio and its components in iron metabolism along with their diagnostic cutoff values, sensitivity, specificity, and accuracy in β-TM patients with iron overload, were examined in this study. This was a 1:1 case-control study with 120 participants, ages ranging from 2 to 30 years of both sexes, who were assigned into two groups: 60 β-TM patients with iron overload, and a control group, comprising 60 healthy individuals matched by gender and age. In the present study, we found slightly elevated serum HEPC concentration (21.9 ng/mL) compared to the controls (9.9 ng/mL), which was not statistically significant (p =0.1), and the median HEPC-to-FER ratio of the cases was significantly lower than the controls, with the median case-control difference of (-0.366; p < 0.001). Our results revealed a statistically significant impact (p < 0.001) of mean age on the serum HEPC level with the inverse linear correlation of (-0.487, p < 0.001). The area under the curve of the HEPC-to-FER ratio was 0.999 and the optimum cutoff value was 0.046 ng/mL (p < 0.001) with 100.0% sensitivity and 98.3% specificity. In conclusion, we found that serum HEPC-to-FER ratio, with an accuracy of 99.2%, may serve as an excellent index for the diagnosis of iron overload in β-TM patients differentiating them from nonthalassemic controls.