The conjunctiva is a highly specialized ocular mucosal surface that, like other mucosa, houses a number of leukocyte populations. These leukocytes have been implicated in age-related inflammatory diseases such as dry-eye, but their phenotypic characteristics remain largely undetermined. Existing literature provides rudimentary data from predominantly immunohistochemical analyses of tissue sections, prohibiting detailed and longitudinal examination of these cells in health and disease. Using recovered cells from ocular surface impression cytology and flow cytometry, we examined the frequency of leukocyte subsets in human conjunctival epithelium and how this alters with age. Of the total CD45+ leukocyte population within the conjunctival epithelium, 87% [32–99] (median) [range] comprised lymphocytes, with 69% [47–90] identified as CD3 + CD56- T cells. In contrast to peripheral blood, the dominant conjunctival epithelial population was TCRαβ + CD8αβ + (80% [37–100]) with only 10% [0-56%] CD4+ cells. Whilst a significant increase in the CD4+ population was seen with age (r = 0.5; p < 0.01) the CD8+ population remained unchanged, resulting in an increase in the CD4:CD8 ratio (r = 0.5;p < 0.01). IFNγ expression was detectable in 18% [14–48] of conjunctival CD4+ T cells and this was significantly higher among older individuals (<35 years, 7[4–39] vs. >65 years, 43[20–145]; p < 0.05). The elevation of CD4+ cells highlights a potentially important age-related alteration in the conjunctival intra-epithelial leukocyte population, which may account for the vulnerability of the aging ocular surface to disease.