Highly sensitive and selective detection of microRNA (miRNA) is becoming more and more important in the discovery, diagnosis, and prognosis of various diseases. Herein, we develop a three-dimensional DNA nanostructure based electrochemical platform for duplicate detection of miRNA amplified by nicking endonuclease. Target miRNA first helps construction of three-way junction structures on the surfaces of gold nanoparticles. After nicking endonuclease-powered cleavage reactions, single-stranded DNAs labeled with electrochemical species are released. These strands can be facilely immobilized at four edges of the irregular triangular prism DNA (iTPDNA) nanostructure via triplex assembly. By evaluating the electrochemical response, target miRNA levels can be determined. In addition, the triplexes can be disassociated by simply changing pH conditions, and the iTPDNA biointerface can be regenerated for duplicate analyses. The developed electrochemical method not only exhibits an excellent prospect in the detection of miRNA but also may inspire the engineering of recyclable biointerfaces for biosensing platforms.