Herein, a novel two-dimensional ladder-like DNA nanostructure (LDN)-mediated cascade catalytic nanomachine (LDN-CCN) with a higher catalytic efficiency beyond those of a conventional one-dimensional hybrid chain reaction (HCR) nanostructure-mediated CCN was constructed and applied to design an electrochemical biosensing platform with first-rank performance for ultrasensitive detection of target miRNA-21. First, output DNA S1' and S2' were produced through the DNAzyme recycle amplification when the target miRNA-21 existed. Then, the controllable LDN-CCN was constructed on S1-S2 modified electrodes by the subsequent reaction triggered by S1' and S2' with H1-AuNPs, H2, H3-AuNPs, and H4 with the assistance of K+ and hemin, in which the hemin/G-quadruplexes could produce a prominent electrochemical signal response to the substrate glucose. The best performance of cascade catalysis was acquired when the distance of Au nanoparticles (glucose oxidase-like activity) modified on H1 and H3 and hemin/G-quadruplexes (peroxidase-like activity) formed by the sticky ends of H2 and H4 was roughly 9 nm (27 bp) in LDN-CCN. The constructed electrochemical platform realized the sensitive detection of target miRNA-21 with the linear range from 100 aM to 10 nM and with a detection limit as low as 48.5 aM, which provided novel insights to explore the new functional DNA nanostructure and well-performing mimic enzyme cascade catalytic platforms for applications in biological fields and early diagnosis of diseases.