A relationship between Alzheimer's disease (AD) and folate has been reported. Amyloid positron emission tomography (PET) is currently one of the most reliable biomarkers for AD. We investigated the correlation between serum folate levels and amyloid imaging to clarify whether serum folate could be a biomarker for AD. We also examined the usefulness of a combined assessment of serum folate levels and red blood cell hemoglobin content. Apolipoprotein E (APOE) gene polymorphisms were also assessed. Serum folate levels and hemoglobin content were evaluated by receiver operating characteristic analysis for their diagnostic capability as AD biomarkers relating to brain amyloid ß accumulation. The area under the ROC curve (AUC) for serum folate was 0.136 (95% confidence interval [CI]: 0.000-0.312; p = 0.016). The AUC for hemoglobin content was 0.848 (95% CI: 0.661-1.000; p = 0.021). Therefore, the folate deficiency with low folate levels or the non-anaemia with high hemoglobin content levels were found to have a high probability of also testing positive for amyloid. Furthermore, eight patients were found to be folate deficiency and non-anaemia, those who were consist of 7 amyloid positive patients (87.5%), and only one of the amyloid negative patients (12.5%). These results suggest that a deficiency of serum folate and high hemoglobin levels may reflect an increased risk of amyloid ß accumulation in the brain. Additionally, we demonstrated that these biomarkers could enhance the effectiveness of APOE as an AD biomarker. This study reveals that the combined assessment of serum folate levels and red blood cell hemoglobin content may be a useful biomarker for amyloid ß accumulation in the brain. We also found that the combination of serum folate levels and hemoglobin content is a more specific and sensitive blood biomarker for AD than APOE or folate alone. These findings may be used to support clinical diagnosis of AD using a simple blood test.