Background: Slit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. Objective: To study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. Methods: Seventy-nine individuals with a mean age of 55.79 {+/-} 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. Results: We found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E (APOE) genotype. Conclusions: SLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.