The survival rates of children with acute lymphoblastic leukemia (ALL) have improved over the years, but infections remain a significant cause of morbidity and mortality. Chemotherapy has a range of harmful side effects including the loss of protective antibodies against vaccine-preventable diseases. The objective of this study was to evaluate the serological status of pediatric ALL cases before and after intensive chemotherapy. Children treated and followed for ALL at Dokuz Eylül University were included in this retrospective cross-sectional study. Antibody levels against hepatitis A, hepatitis B, and rubella were routinely assessed at both the time of diagnosis and 6 months after completion of chemotherapy. Measles, mumps, and varicella antibody levels were evaluated at only 6 months after treatment. Seventy-eight children who completed chemotherapy for ALL were enrolled in the study. All participants had non-protective antibody levels for at least one of the diseases. The highest seropositivity rate was found for hepatitis A (55.1%) and the lowest for measles (17.9%) after chemotherapy. Overall, 50.7%, 30.6%, and 45.7% of the patients significantly lost their humoral immunity against hepatitis B, hepatitis A, and rubella, respectively. Patients in the higher-risk group for ALL had lower seropositivity rates than patients of the other risk groups. There were statistically significant relationships between the protective antibody rates for hepatitis A and varicella and the ages of the patients. Except for hepatitis A vaccination, pre-chemotherapy vaccination did not affect post-chemotherapy serology. On the other hand, all children with a history of varicella before diagnosis showed immunity after chemotherapy. Patients with ALL, including those previously fully vaccinated, are at great risk of infection due to the decrease in protective antibody levels after chemotherapy. There is a need for routine post-chemotherapy serological testing and re-vaccination based on the results obtained.