Plasma miRNAs can characterize several diseases, including acute ischemic stroke (AIS), which is noninvasive and currently affordable in most laboratories worldwide. We aimed to demonstrate plasma miR‐140‐3p, miR‐130a‐3p, and miR‐320b as diagnostic biomarkers in AIS.GSE110993 and GSE86291 datasets were analyzed to obtain plasma differentially expressed miRNAs between AIS and healthy control subjects (HCs). We further applied RT‐qPCR for the validation in 85 AIS patients and 85 HCs. Receiver operating characteristic (ROC) curve were conducted to evaluate their diagnostic utility in AIS. Correlation was analyzed between DEmiRNAs and clinical and laboratory parameters, as well as inflammatory markers. The plasma levels of miR‐140‐3p, miR‐130a‐3p, and miR‐320b were found to be consistently altered in both GSE110993 and GSE86291 datasets. In comparison to HCs, AIS patients at admission exhibited lower levels of miR‐140‐3p and miR‐320b and higher level of miR‐130a‐3p in their plasma. The ROC analysis revealed that plasma miR‐140‐3p, miR‐130a‐3p, and miR‐320b had area under the curve values of 0.790, 0.831, and 0.907, respectively. When combined, these miRNAs showed superior discriminatory power with a sensitivity of 91.76% and specificity of 95.29%. Plasma miR‐140‐3p and miR‐320b negatively correlated glucose levels and inflammatory markers (IL‐6, MMP‐2, MMP‐9, and VEGF) in AIS patients. Conversely, plasma miR‐130a‐3p levels were positively associated with glucose levels and these markers. Plasma miR‐140‐3p, miR‐130a‐3p, and miR‐320b levels varied significantly among AIS patients with different NIHSS scores. Plasma miR‐140‐3p, miR‐130a‐3p, and miR‐320b had high diagnostic value in AIS patients, which were correlated with inflammation and severity in stroke.