Objective: The propose of study was to determine the viral load of Parvovirus B19 in children with proven acute lymphoblastic leukemia (ALL) Methods: A cross-sectional study designed in pediatric ward of Rasoul-e-Akram Hospital (educational, third referral) in Tehran, Iran (2015 – 2016). Sixty-nine children (mean age: 6.57 ± 4.25 years) in preservative phase of ALL selected. BMA had done, and the viral load for Parvovirus B19 determined by quantitative PCR. Results: Type of ALL was Pre B cell: 76.8%; Pro B cell: 14.5% and T-cell type: 7.8%. Low viral load determined in 18.8% (n=13); intermediate in 39.1% (n=27). High and very high viral load observed in 27.1% 15 (n=15) and 20.3% (n=1) of cases respectively. Viral load was related to Pro B cell type (p = 0.050) and lymphadenopathy (p = 0.040), but not related to gender (p = 0.350) and severity of ALL (p = 0.403); like hepatosplenomegaly, anemia, thrombocytopenia or need to transfusion. Conclusion: Parvovirus B19 is a common viral infection in young Iranian population. Near one-third of childbearing age, women in Iran are at risk for primary Parvovirus B19 infection. Here, to evaluate the possible role of parvovirus B19 Infection in the etiology of ALL, 42 % of ALL cases had high and very high viral load (21.7% and 20.3); the low or intermediate viral load is detectable in all cases. The viral load was related to Pro B cell type of ALL (p = 0.050) and the presence of lymphadenopathy. However, there is no relationship observed between Parvovirus B19 viral load and the severity of ALL and gender. It probably might be due to viral detection in the early stages of the ALL