A dual-biomarker, self-powered biosensor was fabricated for the ultrasensitive detection of microRNA-21 (miRNA-21) and miRNA-155 based on enzymatic biofuel cells (EBFCs), catalytic hairpin assembly (CHA), and DNA hybridization chain reaction (HCR), with a capacitor and digital multimeter (DMM). In the presence of miRNA-21, the CHA and HCR are triggered and lead to the generation of a double-helix chain, which stimulates [Ru(NH3)6]3+ to move to the biocathode surface due to electrostatic interaction. Subsequently, the biocathode obtains electrons from the bioanode and reduces [Ru(NH3)6]3+ to [Ru(NH3)6]2+, which significantly increases the open-circuit voltage (E1OCV). When miRNA-155 is present, CHA and HCR cannot be completed, resulting in a low E2OCV. The self-powered biosensor allows for the simultaneous ultrasensitive detection of miRNA-21 and miRNA-155 with detection limits of 0.15 and 0.66 fM, respectively. Moreover, this self-powered biosensor exhibits the highly sensitive detection for miRNA-21 and miRNA-155 assay in human serum samples.