A self-powered microRNA biosensor with triple signal amplification systems was assembled through the integration of three-dimensional DNA walkers, enzymatic biofuel cells and a capacitor. The DNA walker is designed from an enzyme-free target triggered catalytic hairpin assembly of modified gold nanoparticles. When triggered by the target microRNA, the DNA walker will move along the catalytic hairpin track, resulting in a payload release of glucose oxidase. The enzymatic biofuel cell contains the glucose oxidase bioanode and a bilirubin oxidase biocathode that bring a dramatic open circuit voltage to realize the self-powered bioassays of microRNA. A capacitor is further coupled with the enzymatic biofuel cell to further amplify the electrochemical signal, and the sensitivity increases 28.82 times through optimizing the matching capacitor. Based on this design, the present biosensor shows high performance, especially for detection limit and sensitivity. Furthermore, the present biosensor was successfully applied for serum samples, directly demonstrating its good application in clinical biomedicine and disease diagnosis.