Abstract <p>A ratiometric electrochemical aptasensor for the sensitive detection of mucin 1 (<b>MUC1</b>) was designed based on a quadruped DNA walker driven by catalytic hairpin assembly (<b>CHA</b>). With the tetrahedral DNA nanostructure as the framework, the four legs that extend out acted as the walking chains. MUC1 bound specifically to the aptamer, activating the quadruped DNA walker and initiating the CHA reaction. The activated quadruped DNA walker hybridized with hairpin DNA1 labeled with methylene blue (<b>MB-HP1</b>) immobilized on the electrode surface, and the toehold area of the MB-HP1 probe was exposed to bind with hairpin DNA2 labeled with ferrocene (<b>Fc-HP2</b>), causing the quadruped DNA walker to be released. It continued to move on the electrode surface and bound with another MB-HP1. Eventually, a large number of electrochemical signal molecules were brought close to the electrode surface, while MB moved farther away. Under optimal conditions, the peak current ratio (<i>I</i>_Fc/<i>I</i>_MB) was linearly correlated with the logarithm of MUC1 concentration from 1 fg/mL to 1 ng/mL, with a detection limit as low as 0.56 fg/mL. In addition, the sensing platform demonstrated good performance in the analysis of serum samples.</p>