<p>Early and economical diagnosis of acute ischemic stroke (AIS) is pivotal for therapeutic efficacy, particularly for the settings where medical imaging resource is deficient. We have obtained evidence supporting our hypothesis that collective properties of the green autofluorescence (AF) of the fingernails and certain skin’s positions may be a novel diagnostic biomarker for AIS: both the green AF intensity and AF asymmetry of the AIS patients in their Index Fingernails and most examined skin’ s positions were significantly higher than that of the healthy subjects and the Non-AIS subjects. Receiver operating characteristic (ROC) analyses and machine learning-based analyses on the AF properties showed that area under curve (AUC) was 0.93 and 0.87, respectively, for differentiating the AIS patients from the healthy subjects and for differentiating the AIS patients from the Non-AIS subjects. The AIS patients had significantly higher AF intensity and AF asymmetry at several examined positions, compared to those of the patients of Parkinson’s disease, pulmonary infection and transient ischemic attack. The AUC was 0.79–0.88 for differentiating AIS patients from each of these diseases. Our study has also obtained evidence implicating that the AF originates from keratins. Collectively, our study has obtained preliminary evidence suggesting that the characteristic AIS’s ‘Pattern of AF’ is a novel diagnostic biomarker for the disease. The ‘Pattern of AF Technology’ holds potential to become a new non-invasive, label-free and economical diagnostic approach for AIS, which is particularly valuable when Magnetic Resonance Imaging (MRI) or Computed Tomography (CT) imaging resource is deficient.</p>