Background: Vitamin B12 and folate are essential micronutrients for normal growth, neurodevelopment, and hematopoiesis in children. However, age-specific data on the prevalence of vitamin B12 and folate deficiency in pediatric populations remain limited. Methods: This cross-sectional study retrospectively analyzed serum vitamin B12 and folate measurements obtained from children and adolescents aged 0–18 years who attended a general pediatrics outpatient clinic at a tertiary care hospital between 2020 and 2025. Participants were stratified into four age groups (0–2, 2–6, 6–12, and 12–18 years). Vitamin B12 deficiency was defined as <200 pg/mL and borderline deficiency as 200–300 pg/mL, while folate deficiency was defined as <5 ng/mL. Age- and sex-specific prevalence rates were evaluated, and annual mean vitamin levels were assessed. Results: A total of 196,091 serum measurements were analyzed over the six-year period. Vitamin B12 deficiency and borderline low vitamin B12 levels were observed across all age groups, with the highest prevalence in children aged 0–2 years and in adolescents. Folate deficiency was uncommon in early childhood but increased markedly during adolescence. Children aged 2–6 years demonstrated the lowest prevalence of both vitamin B12 and folate deficiency. No significant differences were observed between sexes. Annual mean serum vitamin B12 levels remained relatively stable, whereas serum folate levels showed notable interannual variability. Conclusion: Vitamin B12 and folate deficiency exhibit distinct age-related patterns during childhood and adolescence. Early childhood and adolescence represent vulnerable periods for vitamin B12 insufficiency, while folate deficiency is primarily an adolescent concern. These findings highlight the importance of age-targeted nutritional surveillance and preventive strategies in pediatric care.