DNA walking machines opened new avenues for the biosensing and demonstrated great success in the past few years. Since DNA machines are mainly nonequilibrium systems driven by dynamic interactions, the matrix effects on DNA machines is a bottleneck and more intricate than common DNA-mediated assays, especially for complicated physiological samples. Herein, to realize an accurate and reliable quantitative machine, a ratiometric DNA walking machine was developed in human serums and cell lysates based on the elemental isotope ratio measurement. The target DNA-triggered walking machine converted and amplified biological signals into mass spectrometric signal ratios (197 Au/115 In) via a burnt-bridge mechanism. Under the optimized conditions, the limit of detection (LOD, 3σ) was 8 fM for target DNA, with a dynamic linear range of 0.05-0.7 pM. The ratiometric DNA walking machine was directly applied in human serum samples with satisfactory recoveries of 94 to 105 %, demonstrating an excellent stability and a high accuracy. Combining the aptamer-based specific recognition, the proposed DNA machine is expected to be a versatile platform for other targets, such as small biomolecules and proteins.