<b><i>Introduction:</i></b> Acute cerebral infarction (ACI) occurs with the involvement of differential expression of microRNAs. The study detected the expression pattern of miR-138-5p in the serum of ACI cases and evaluated its clinical significance, in an attempt to provide some guidance for the treatment and daily nursing of patients with ACI clinically. <b><i>Methods:</i></b> Levels of miR-138-5p in the serum of ACI patients and healthy controls (HCs) were detected via qRT-PCR. Ninety days after treatment, the modified Rankin Scale (mRS) was used to evaluate the prognosis of ACI patients. Receiver operating characteristic (ROC) curve was drawn, and the area under the curve was calculated. The logistic regression analysis was performed to estimate the relationship between various indicators and the clinical outcome. <b><i>Results:</i></b> miR-138-5p showed a diminished trend in ACI cases compared with the control group. A significantly negative correlation was detected for serum miR-138-5p with the National Institutes of Health Stroke Scale score in all ACI cases (<i>r</i> = −0.704, <i>p</i> &#x3c; 0.001). The ROC curve demonstrated the diagnostic potential of serum miR-138-5p to distinguish ACI from HCs. Lessened expression of miR-138-5p was detected in ACI patients with poor prognosis, which can predict the poor prognosis of ACI patients after treatment. Logistic regression analysis determined the independent influence relationship between miR-138-5p and poor prognosis. <b><i>Conclusion:</i></b> Diminished miR-138-5p is identified to be a risk factor for the occurrence of ACI, and it is associated with the worse outcome of the patients.