Purpose <p>The early diagnosis of malignant transformation in oral leukoplakia (OLK) assumes paramount importance in the preventive and timely intervention strategies for oral cancer. Nevertheless, the existing diagnostic modalities exhibit complexity, invasiveness, or inherent limitations. The therapeutic efficacy and mechanism of miR-185 in extracellular vesicles (EVs) in oral cancer have been previously demonstrated. Thus, this study aims to establish a diagnostic model using miR-185 from salivary EVs to identify the malignant transformation of OLK and facilitate the prevention and treatment of oral cancer.</p> Methods <p>miR-185 in salivary EVs was characterized in a discovery cohort comprising healthy controls (<i>n</i> = 8) and patients with hyperplasia (<i>n</i> = 13), mild to moderate dysplasia (<i>n</i> = 15), severe dysplasia (<i>n</i> = 10), and squamous cell carcinoma (OSCC, <i>n</i> = 23). A training set from a validation cohort (<i>n</i> = 164) underwent analysis using receiver operating characteristic curve (ROC) and logistic regression analysis to distinguish malignant forms (MT; i.e., severe dysplasia and OSCC) from non-malignant forms (N-MT; i.e., hyperplasia, mild to moderate dysplasia). The validation set was then utilized to validate the model.</p> Results <p>miR-185 in salivary EVs demonstrated accurate identification of MT [area under ROC (AUC) = 0.9349, specificity = 90.32%, sensitivity = 80.77%], and its feasibility and accuracy were successfully confirmed in the validation set (specificity = 93.55%, sensitivity = 84.21%).</p> Conclusion <p>miR-185 in salivary EVs can serve as a non-invasive biomarker for the detection of OLK malignant transformation.</p>