The aim of this study was to assess serum ferritin, hepcidin, L-fucose, and protein binding fucose levels in β-thalassemia major (β-TM) patients and to correlate the serum ferritin level with hepcidin and fucose levels. A total 70 (26 males and 44 females) children with β-TM, ages ranging from 5 to 16 years (mean age 8.3 ± 2.7 years) and 50 (25 males and 25 females) apparently healthy subjects with matching age and sex were included as a control group. An especially designed questionnaire was used to collect age, gender, body mass index (BMI), hemoglobin (Hb), serum ferritin, hepcidin-25 peptide, α-L-fucose, and protein binding fucose (PBF) levels. β-Thalassemia major patients had significantly (p < 0.05) higher serum ferritin, fucose and PBF levels, but the serum hepcidin level was significantly (p < 0.05) lower when compared to the controls, and their levels were affected by the gender of the β-TM patients, as it was significantly (p < 0.05) higher in female in comparison to male patients. There was no significant (p > 0.05) correlation between serum ferritin with hepcidin and fucose levels as a marker of iron overload in β-TM. The regulation of hepcidin, and L-fucose levels in patients with β-TM is more affected by erythropoeitic activity than by iron overload, as there was no significant correlation between serum ferritin with hepcidin and fucose levels as a marker of iron overload in β-TM.