Abstract Background: The role of immune system in natural course of viral hepatitis has been drawn some attention. One of the main diagnostic markers of the immune system function in different diseases might be the ratio of CD4+ to CD8+ T lymphocytes (CD4+/CD8+ ratio). Objectives: This research aimed tomeasure and compare CD4+/CD8+ ratio in the patientswith chronic hepatitis B(CHB) and control group. Methods: In this cross-sectional study, thirty-three CHB patients and thirty age and sex-matched healthy controls were included. Immunophenotyping of isolated T cells was performed using specific anti-CD4 and anti-CD8 antibodies by flow cytometry. Consequently, CD4+, CD8+ and the ratio of CD4+/CD8+were counted and compared between the two groups. Results: CD4+ counts (%) were considerably reduced in patients with CHB compared to the healthy controls (51.22 ± 10.5 vs. 63.14 ± 9.9, P = 0.00), whereas CD8+ counts (%) were higher in the patients with CHB than healthy controls (48.8 ± 10.5 vs. 36.85 ± 9.86, P = 0.00). Moreover, CD4+/CD8+ ratio remarkably decreased in the patients with CHB (1.15 ± 0.5) than healthy controls (1.93 ± 0.9) (P = 0.00). Area under curve (AUC) of 0.79 (SE = 0.06, CI = 0.68 - 0.90, P value = 0.05) was reported for CD4+/CD8+ ratio with a sensitivity of 72.73% and specificity of 73.33% in 1.35 cut-off (likelihood ratio = 2.72). Conclusions: The research indicated an impaired balance between T cell subsets associated with a higher proportion of CD8+ T cells and a lower proportion of CD4+ T cells and CD4+/CD8+ ratio in patients with CHB