The epsilon 4 allele of the APOE gene is thought to increase risk from amnestic mild cognitive impairment (aMCI) to Alzheimer's disease. Cognitive decline in the condition is increasingly considered to worsen functional disconnections in brain network composed of gray matter and white matter. Nevertheless, Whether APOE epsilon 4 targets specific white matter functional connectivity in patients with aMCI remains mostly unexplored, mainly due to the challenges of detecting BOLD signals in white matter. Here, we applied a novel approach to investigate APOE epsilon 4-related specific bundles and cortical area alterations in aMCI subjects, in order to characterize white matter-gray matter functional connectivity differences throughout the brain. We analyzed 75 patients with aMCI and 76 demographically matched normal controls. The aMCI APOE epsilon 4 carriers showed decreased functional connectivity located at left corticospinal tract, bilateral posterior limb of internal capsule, and right temporopolaris, which was different from the regions of aMCI-related changes. We further found that recognition scores were positively associated with the right temporopolaris in aMCI APOE epsilon 4 carriers. Collectively, the data provide new evidence that APOE epsilon 4 genotype exerts a negative impact on neural activity in both gray and white matter in aMCI, which potentially contributes to functional disconnection and memory decline. A novel method provides full-scale measuring effect of disease conditions on functional architecture throughout the brain. Trial registration:(Identifier: NCT02225964). Registered January 2014.