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To determine region-specific reference ranges of lymphocyte T subsets in blood donors and to assess the influence of gender and age on lymphocyte T susbsets.Blood samples from 143 blood donors were collected in the Blood Transfusion Center of Sfax. Lymphocyte T subsets were analyzed using a dual-platform method with a flow cytometer (percentages) and an automated hematology analyzer (white blood cells and lymphocytes). ANOVA and Student's tests were used for data analysis.Reference values were expressed as mean and 95% confidence intervals for T cells: CD3+: 1415 ± 348 cells/μL [1357-1473], CD3+/CD4+: 786 ± 220 cells/μL [732.31-811.7], CD3+/CD8+: 639 ± 258 cells/μL [596-862] and CD4+/CD8+ ratio was 1.46 ± 0.77 [1.36-1.62]. Gender and age influenced blood lymphocyte T subsets.Our study leads to the establishment of peripheral blood lymphocyte T subset reference ranges in blood donors in the region of Sfax. A study on a more diversified population, including more important number of individuals from various regions of Tunisia and including enumeration of other lymphocyte subsets (B cells and NK cells) is required.