The ultrasensitive electrochemical detection of miRNA-21 was realized by using a novel redox and catalytic “all-in-one” mechanism with an iridium(III) complex as a catalyst. To construct such a sensor, a capture probe (CP) was firstly immobilized onto the gold electrode surface. In the presence of miRNA-21, a sandwiched DNA complex could form between CP and a methylene blue (MB) labeled G-rich detection probe modified onto a gold nanoparticle (AuNP) surface (DP-AuNPs). Upon addition of K+, the structure of DP changed to a G-quadruplex. Then, the iridium(III) complex could selectively interact with the G-quadruplex, catalyzing the reduction of H2O2, which was accompanied by an electrochemical signal change using MB as an electron mediator. Under optimal conditions, the electrochemical signal of MB reduction peak was proportional to miRNA concentration in the range from 5.0 fM to 1.0 pM, with a detection limit of 1.6 fM. In addition, satisfactory results were obtained for miRNA-21 detection in human serum samples, indicating a potential application of the sensor for bioanalysis. © 2016 Elsevier B.V.