<p>Alzheimer’s disease (AD) is the most common form of dementia. However, there is a lack of effective diagnostic biomarkers to differentiate AD from non-Alzheimer’s (Non-AD) dementias, such as vascular dementia, Lewy body dementia, Parkinson’s disease dementia, and chronic traumatic encephalopathy. This study aims to evaluate the expression variations of LINE-1 retrotransposon in plasma EVs as a potential biomarker to differentiate between AD and Non-AD dementias and explore its diagnostic value in clinical practice. Here, we analyzed two independent clinical cohorts: a training cohort (AD: <i>n</i> = 139; Non-AD: <i>n</i> = 137; Control: <i>n</i> = 79) and a validation cohort (AD: <i>n</i> = 158; Non-AD: <i>n</i> = 133; Control: <i>n</i> = 61). Expression of LINE-1 mRNA in plasma EVs, containing ORF1, ORF2, and 5’UTR regions, was quantified using RT-qPCR, and the differences between control, AD and Non-AD groups were assessed by one-way ANOVA or non-parametric tests. In both training and validation cohorts, LINE-1 mRNA expression was significantly higher in AD patients compared to Non-AD patients and healthy control in the ORF1, 5’UTR, and ORF2 regions. LINE-1 expression was correlated with that of neuroinflammatory and neurodegeneration markers (GFAP and NfL), whereas no significant correlations were revealed between LINE-1 expression and cognitive scores. ROC analysis revealed that while LINE-1 was less effective than traditional biomarkers for general AD diagnosis, it demonstrated superior accuracy in differentiating AD from Non-AD dementias. Specifically, the AUC values for LINE-1_ORF2 and LINE-1_5’UTR were 0.91 and 0.92, respectively, significantly outperforming p-Tau217 (AUC = 0.80) and GFAP (AUC = 0.62) in the training cohort. Multivariate regression confirmed that the LINE-1 composite index was an independent predictor of AD. These findings suggest that the LINE-1 composite score, as an age-independent predictor, holds significant clinical potential for the differential diagnosis of AD.</p>