Background: Beta thalassemia major (β-TM) is an inheritable condition with many complications, especially in children. The blood-borne viral infection was proposed as a risk factor due to the recurrent blood transfusion regimen (hemotherapy) as human parvovirus B19 (B19V). Objective: This study investigated the B19V seroprevalence, DNA presence, B19V viral load, and B19V genotypes in β-TM patients and blood donors. Methods: This is a cross-sectional study incorporating 180 subjects, segregated into three distinct groups each of 60 patients, namely control, β-TM, and β-TM infected with Hepatitis C Virus (HCV).  For the B19V prevalence in the studied group, the ELISA technique and real-time PCR were used. The genotyping was followed by the resultant sequence. Results: Both B19V IgM and IgG antibody positivity rates are higher among β-TM patients compared to controls. The B19V IgM (35%) and B19V IgG (21.67%) antibodies positivity in β-TM patients compared to 23.3% and 18.33% positivity in the controls was significantly observed. The prevalence of B19V was (8.3%), and the viral copy number in β-TM patients ranged from ≥104– 106 copies/ml than in controls. The B19V genotype 1 subtype a was the only genotype according to the VP1-VP2 region (288 pb) in this study. Conclusions: The prevalence of B19V in patients may be higher than in controls. B19V screening in high-risk groups, such as blood donors, may considerably reduce the prevalence of B19V.