Objective: The pathophysiology of attention deficit hyperactivity disorder (ADHD) are still not fully elucidated. Immune system dysregulation has emerged as a major etiological focus as a result of the high comorbidity of allergic disease, inflammatory biomarkers, and genetic research. The present study aimed to evaluate peripheral lymphocyte subpopulations and regulatory T cells (Tregs) in children with ADHD. Methods: This single-center cross-sectional case-control study assessed 49 children with ADHD and 35 age- and gender-matched healthy children aged 7–12 years (9.10 ± 2.37 and 9.45 ± 2.13, respectively). The participants were screened for psychopathology using the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children–Present and Lifetime Version, while the severity of ADHD symptoms was measured by means of the distracted-Continuous Performance Test. Peripheral lymphocyte subpopulations and Tregs were analyzed with flow-cytometry. Results: There is no significant difference in peripheral blood lymphocyte subsets between ADHD and control groups The children diagnosed with ADHD exhibited significantly higher levels of CD3+ CD4+ CD25+ Foxp3+ (Tregs) than the healthy control subjects (8.23 ± 2.09 vs. 6.61 ± 2.89; z = 2.965, p = .004). The Tregs cell (Exp(B) = 1.334; p = .042; CI = 1.011–1.761) levels were determined to be statistically significant according to regression analysis and were associated with an increased probability of ADHD. Conclusion: Elevated Treg levels were linked to an increased likelihood of ADHD. This study suggested that changes in immune regulatory cells represent an important part of research in treatment of ADHD.