Aim To explore the association and role of serum miR-335 in patients with acute ischemic stroke（AIS）. Methods Blood samples were obtained from AIS patients （n106） and healthy controls （n98）. miR-335 was measured by using a quantitative real-time PCR technique. Stroke severity was evaluated by the National Institutes of Health Stroke Scale（NIHSS）. The correlation between serum miR-335 and NIHSS scores was performed by Pearson correlations analysis. Bioinformatics database assay was used to predict comprehensively target genes, Gene ontology and enriched pathway of target genes were analyzed with DAVID database. Results Compared with healthy controls, serum miR-335 was significantly decreased in stroke patients （P<0.01）, especially at 3 days（P<0.01）. The level of serum miR-335 was significantly lower in NIHSS > 5 group compared with NIHSS≤5 group（P<0.01）, the level of miR-335 was closely negatively correlated to NIHSS（r-0.28,P<0.01）. The predicted target genes of miR-335 were mainly involved in biological processes including metabolism of nucleic acid, regulation of transcription, growth and differentiation of neurons, angiogenesis, molecular function including regulation of channel activity, calcium ion binding activity, cationic metal ion binding activity and transcriptional activity, and cellular components including nucleoplasm, pathways including oxidative stress response, angiogenesis and MAPK signaling pathway. Conclusions The level of miR-335 was significantly reduced in serum of patients with acute ischemic stroke, and serum miR-335 may be a novel sensitive biomarker for clinical diagnosis in acute cerebral ischemia.