G-quadruplex/hemin (G4/hemin) DNAzymes are biosensing systems, but their application remains limited by an overall low activity and a rather high level of unwarranted background reactions. Here, these issues were addressed through the rational design of F3T-azaC-hemin, a G4-based construct in which the hemin is covalently linked to the G4 core and its binding site flanked with a nucleotide activator, here d(T-azaC). This design led to a G4-DNAzyme whose performances have been ca. 150-fold increased compared to the parent G4-based system. The utility of F3T-azaC-hemin was demonstrated here through the ultrasensitive chemiluminescent detection of miRNA-221. The limit of detection (LOD) has been decreased to the femtomolar range, making it a new and highly efficient molecular tool in the biosensing technology field.