Aging is a critical global issue that contributes to the high incidence of Alzheimer's disease (AD). Blood screening emerges as the most promising measure for early diagnosis and intervention of AD due to its noninvasive and low cost. However, the practical application of AD blood screening confronts two significant challenges. First, due to the blood−brain barrier, the concentration of AD biomarkers in blood is much lower than that in cerebrospinal fluid. Second, simultaneous quantitative analysis of multiple biomarkers is necessary due to the low specificity of individual biomarkers. Herein, we propose DNAzyme-based 3D DNA walkers for the sensitive and multiplex detection of five ADassociated miRNA biomarkers: hsa-miR-125b, hsa-miR-342-3p, hsa-miR-29b, hsa-miR-191-5p, and hsa-miR-7d-5. The DNAzymebased 3D DNA walkers provide highly efficient and autonomous amplification of the minimal biomarkers' quantities. The walkingreleased metal isotopes 89 Y, 165 Ho, 139 La, 140 Ce, and 159 Tb can be sensitively detected by elemental mass spectrometry without any spectral overlap. The detection limit was achieved to be as low as 1.0 fmol. The proposed method was successfully applied to human serum samples with satisfactory spiked recoveries. With its high sensitivity and multiplexity capabilities, this metal isotope strategy may contribute to the early diagnosis and intervention of AD.