We have previously shown the neuroprotective function of microRNA-99a-5p in experimental stroke. Here, we explore its diagnostic potential for acute stroke patients. MiR-99a-5p levels in circulating from acute stroke patients and control were measured by real-time polymerase chain reaction. Pearson's correlation and receiver operator characteristic (ROC) curves were used to analyze clinical significance of miR-99a-5p and its sensitivity and specificity for stroke diagnosis. We demonstrated that miR-99a-5p expression was upregulated in neutrophils of both ischemic stroke and hemorrhage patients, while was only increased in the lymphocytes of hemorrhage patients. ROC analysis revealed that the miR-99a-5p level in neutrophils and lymphocytes had a moderate diagnostic value for stroke. Moreover, a positive correlation existed between plasma miR-99a-5p levels and neutrophil numbers or neutrophil/lymphocyte ratio. Meanwhile, miR-99a-5p levels in neutrophils were negatively correlated with thrombin time, while positively correlated with D-dimer and urea levels. Lymphocytic miR-99a-5p levels were positively correlated with platelet mean volume and distribution width. This study demonstrated that miR-99a-5p levels in circulating immune cells might facilitate the diagnosis of ischemic stroke.