Changes in epigenetic marks may help explain the late onset of Alzheimer’s disease (AD). In this study we measured genome-wide DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA isolated from peripheral blood mononuclear cells of 37 subjects with late-onset AD (LOAD) and 44 healthy controls (CT). We found an increase in global DNA methylation in LOAD subjects compared to CT (p = 0.0122), associated with worse cognitive performances (p = 0.0002). DNA hypermethylation in LOAD group was paralleled by higher DNA methyltransferase 1 (DNMT1) gene expression and protein levels. When data were stratified on the basis of the APOE polymorphisms, higher DNA methylation levels were associated with the presence of APOE ε4 allele (p = 0.0043) in the global population. Among the APOE ε3 carriers, a significant increase of DNA methylation was still observed in LOAD patients compared to healthy controls (p = 0.05). Our data suggest global DNA methylation in peripheral samples as a useful marker for screening individuals at risk of developing AD.