The electrochemical aptamer sensor has been designed for detecting tau381, a critical biomarker of Alzheimer′s disease in human serum. The aptasensor is obtained by immobilizing the aptamer on a carboxyl graphene/thionin/gold nanoparticle modified glassy-carbon electrode. As a probe and bridge molecule, thionin connected carboxyl graphene and gold nanoparticles, and gave the electrical signal. Under optimal conditions, the increment of differential pulse voltammetry signal increased linearly with the logarithm of tau381 concentration in the range from 1.0 pM to 100 pM, and limit of detection was 0.70 pM. The aptasensor reliability was evaluated by determining its selectivity, reproducibility, stability, detection limit, and recovery. Performance analysis of the tau381 aptasensor in 10 patients’ serum samples showed that the aptasensor could screen patients with and without Alzheimer′s disease. The proposed aptasensor has potential for use in clinically diagnosing Alzheimer′s disease in the early stage.