Objective To explore dynamic changes of peripheral blood lymphocyte subsets in patients with acute ischemic stroke (AIS) and the relationship with stroke severity and long-term outcomes. Methods A total of 96 consecutive patients with AIS and 28 age- and gender-matched healthy controls were recruited. Peripheral blood samples were collected, and the percentages of lymphocyte subsets were analyzed by flow cytometry. The dynamic changes in lymphocyte subsets and their correlation with clinical parameters, such as National Institutes of Health Stroke Scale (NIHSS) scores at onset and modified Rankin scale (mRS) scores 3 months later, were evaluated. Results In our study, we observed a decrease in the percentages of T-lymphocytes (T cells), helper/inducible T-lymphocytes (Th cells) and suppressor/cytotoxic T-lymphocytes (Ts cells) in AIS patients as compared to controls. The frequencies of T cells and Ts cells on day 8-14 after stroke in NIHSS <= 4 group were significantly higher than those in NIHSS >4 group. The percentages of T cells and Th cells on day 1-3 after stroke in the mRS <= 2 group were higher than those in the mRS >2 group. Conclusion The frequencies of T cells, Th cells, and Ts cells in AIS are declined dramatically at least 14 days after stroke. Lower frequencies of T cells and Ts cells on day 8-14 after stroke represent more severe disease conditions, and the percentages of T cells and Th cells within 72 hr after stroke are negatively correlated with 3-month outcomes, which might have a potential for predicting long-term prognosis of stroke.