<p>Aspartate, a key excitatory neurotransmitter, has shown conflicting links to cognitive disorders like Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Given its role in brain function, studying its relationship with amyloid-beta (Aβ) accumulation may provide important insights into these conditions. This study is the first to examine the link between serum aspartate levels, Aβ accumulation in key brain regions, and cognitive function (via ADAS-Cog) across cognitively normal (<i>n</i> = 113), MCI (<i>n</i> = 283), and AD (<i>n</i> = 24) participants. The results showed significant increases in Aβ accumulation across all brain regions from CN to MCI and AD groups (overall <i>p</i> &lt; 0.001; pairwise <i>p</i> ≤ 0.001). In individuals with MCI, elevated aspartate levels were inversely associated with Aβ accumulation in the frontal (<i>β</i> = − 0.122, <i>p</i> = 0.041) and temporal regions (<i>β</i> = − 0.128, <i>p</i> = 0.032), but this relationship was lost after adjusting for age, gender, education, handedness, and ApoE ɛ4, ɛ3, and ɛ2 genotypes. Further analysis identified age and ApoE ɛ4, ɛ3 status as key modifiers of the aspartate–Aβ relationship in MCI. Mediation analysis revealed that higher serum aspartate levels were associated with better cognitive function, potentially mediated by reduced Aβ accumulation in the frontal (<i>β</i> = − 0.037) and temporal lobes (<i>β</i> = − 0.043) in MCI, but this effect disappeared after adjusting for demographic factors and ApoE genotypes. Overall, higher serum aspartate levels may be associated with reduced Aβ accumulation and improved cognitive outcomes in MCI, with age and ApoE genotypes acting as key confounders.</p>