Herein, a dual microRNA (miRNA) powered bi-directional DNA walking machine with precise control was developed to fabricate an enzyme-free biosensor on the basis of distance-based electrochemiluminescence (ECL) energy transfer for multiple detection of miRNAs. By using miRNA-21 as the driving force, the DNA walker could move forth along the track and generated quenching of ECL response due to the proximity between Au nanoparticles (AuNPs) and Mn2+ doped CdS nanocrystals (CdS:Mn NCs) film as the ECL emitters, realizing ultrasensitive determination of miRNA-21. Impressively, once miRNA-155 was introduced as the driving force, the walker could move back along the track automatically, and surface plasmon resonance (SPR) occurred owing to the appropriate large separation between AuNPs and CdS:Mn NCs, achieving an ECL enhancement and realizing ultrasensitive detection of miRNA-155. The bi-directional movement of the DNA walker on the track led to continuous distance-based energy transfer from CdS:Mn NCs film by AuNPs, which resulted in significant ECL signal variation of CdS:Mn NCs for multiple detection of miRNA-21 and miRNA-155 down to 1.51 fM and 1.67 fM, respectively. Amazingly, the elaborated biosensor provided a new chance for constructing controllable molecular nanomachines in biosensing, disease diagnosis, and clinical analysis.