Background: β-Thalassemia major (β-TM) is one of the most frequent monogenic inherited hemoglobin disorders worldwide, characterized by absent or severely reduced β-globin chain production, resulting in an imbalanced α/β chain ratio, ineffective erythropoiesis, and chronic hemolytic anemia. To evaluate immune-inflammatory interactions with β-TM pathogenesis following splenectomy by quantifying complement regulatory protein expression (CD55, CD59, CD35) on erythrocytes, and to determine their diagnostic utility in distinguishing splenectomized from non-splenectomized patients and from healthy controls. Methods: A case-control study conducted at Zagazig University Hospitals enrolling 57 participants in three age- and sex-matched groups of 19 each: β-TM patients with prior splenectomy, β-TM patients without splenectomy, and healthy controls. Flow cytometric assessment of CD55, CD59 (on granulocytes), and CD35 and CD59 (on erythrocytes) was performed. Hematological, hepatic, and biochemical parameters were compared, and ROC curve analysis was used to determine diagnostic performance. Results: Splenectomized patients had significantly reduced transfusion requirements post-operatively. Post-splenectomy hematological changes included marked leukocytosis and thrombocytosis. A graded reduction in CD55, CD59, and CD35 was observed from controls to non-splenectomized patients to splenectomized patients. Reduced complement regulatory proteins correlated inversely with inflammatory markers and ferritin. For discriminating splenectomized from non-splenectomized patients, CD35 showed the highest diagnostic accuracy. For discriminating all β-TM patients from controls, CD59 showed the best performance. Conclusion: Splenectomy worsens complement dysregulation in β-TM, evidenced by significantly lower erythrocyte complement regulatory protein expression. CD35 is the most accurate single marker for identifying the post-splenectomy state, while CD59 best distinguishes β-TM patients from healthy individuals.