In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.