A label-free electrochemical biosensor for ultrasensitive let-7d RNA detection was developed based on nicking endonuclease (NEase) assisted template enhanced hybridization process (TEHP) and rolling circle amplification (RCA) induced formation of G-quadruplex-hemin complexes. The hairpin probe (H1) was immobilized on the gold electrode through a strong Au-S bond. With the help of assistant DNA, the introduction of target let-7d RNA forms the nicking site for the NEase, which cleaves the hairpin probe and releases the target RNA. Further addition of another hairpin probe (H2) initiated the next two cascaded recycling process. The cleaved fragment acted as primer to activate RCA reaction, generating product containing hundreds of hemin aptamer and G-quadruplex-hemin complex can be formed with the help of K+ and hemin to give an electrochemical response. The newly designed protocol provides an ultrasensitive electrochemical detection of let-7d RNA down to the 0.42 fM level, and can discriminate target RNA from the let-7 family, holding a great potential for early diagnosis in gene-related diseases. (C) 2016 Elsevier B.V. All rights reserved.