Alzheimer's disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid-β (Aβ) plaques and intracellular tau tangles. This study investigated amyloid beta (Aβ) and tau biomarkers in tear fluid-derived extracellular vesicles (EVs). Tear fluid samples were collected using Schirmer's strips from cognitively impaired (n = 20) and cognitively normal (n = 10) study subjects. EVs were isolated using ExoQuick, characterized by nanoparticle tracking analysis, and lysed with RIPA buffer. The total protein content was quantified using a bicinchoninic acid assay. Immunoassays were used to measure phosphorylated tau 181 (pTau-181), Aβ38, Aβ40, and Aβ42 in both EV-bound and unbound protein fractions. Significantly higher (1.7-fold) levels of pTau-181 were detected as EV-bound proteins as compared to unbound proteins (P = 0.016), suggesting selective packaging of tau into EVs. In contrast, over 88 % of all Aβ isoforms were found as unbound proteins, with lower presence as EV-bound. Additionally, the total protein levels in EVs were significantly higher in the cognitive impaired group compared to the cognitively normal group (2.8-fold). These results demonstrate the presence of AD biomarkers in tear fluid-derived EVs, their association with cognitive impairment and the importance of EV isolation for the improved detection of pTau-181 proteins. Tear fluid-derived EV AD biomarkers may be a promising avenue for non-invasive diagnosis of AD.