Introduction: Regular blood transfusion for β-thalassemia patients is a life-saving therapy, hence, it results in iron overload lead to immune dysregulation triggered by chronic activation of immune system. This fundamental notion contributes to their morbidity and mortality. Monocyte plays a critical role in regulating and bridging innate to adaptive immunity. Our pilot study analyzed the presence of activation markers, CD14 and CD69, on monocyte of major β-thalassemia patients associated with their iron status. Method: Fifty pediatric β-thalassemia patients routinely visited thalassemia clinic for clinical examination and blood transfusion were involved in this cross-sectional study. Flow cytometry applying antibody of CD14, HLA-DR, CD69 was used to dissect CD14+CD69+ monocytes from lysed-erythrocyte heparinized whole blood and defined as cell percentage also median fluorescent intensity (MFI) of CD69 of CD14+CD69+ monocytes. Iron status was indicated by ferritin and serum iron level. A correlation study was done. Results : We found 87.4% (76.1 – 91.4) CD14+CD69+ of dissected monocytes from iron overloaded pediatric β-thalassemia patients (Ferritin level: 3118 μg/L, 1675 – 9718). Positive correlation was found between percentage of CD14+CD69+ monocytes and ferritin level (r = 0.3, P = 0.04). Conclusion: Considering the function of CD14 and CD69 on monocyte and the iron accumulation, our result may implicate that pediatric major β-thalassemia patients have a tendency towards chronic inflammation. Future direction for research of our study aimed at discovering collateral activation of immune cells via monocyte to explain organ damage caused by iron overload is imperative.