Introduction: Isolation of specific cell types is important in providing a better understanding of hematological disorders. The knowledge of molecular biology aspect in β-thalassemia is still limited. This is because hemoglobin disorder involves various erythropoietic processes in which the genetic information is lack due to enucleation of red blood cells occurs in bone marrow. It is invasive to collect samples from bone marrow and cord blood although nucleated red blood cells (NRBCs) are abundant in these sites. NRBCs are precursors of red blood cells and typically found in peripheral blood (PB) of β-thalassemia major patients and abundant post-splenectomy. The utilization of PB NRBCs will provide a further understanding of the molecular aspects of ineffective erythropoiesis in β-thalassemia major patients. Objective: The objective of this study was to isolate the NRBCs using CD71 magnetic beads from PB of β-thalassemia major; non-splenectomy and post-splenectomy patients. Methods: NRBCs were isolated from 6 mL PB of β-thalassemia major patients based on density gradient and magnetic activated cell sorting (MACS) for NRBCs enrichment using a CD71 marker. Cell count was determined by using hemocytometer (Weber Scientific, NJ, USA) and BD FACSCantoTM II flow cytometry (Becton-Dickson, NJ, USA) was performed for method validation. Results: NRBCs were successfully isolated from the PB of both non-splenectomy and post-splenectomy β-thalassemia major patients with >90% specificity by flow cytometric analysis. The median number of enriched NRBCs (x104 ) was 58.5 (283) and 340 (338) respectively using hemocytometer. Conclusion: The MACS method was found to be convenient and efficient in the isolation of the targeted cells for downstream applications.