AbstractIn this study, a hierarchically porous Zr‐MOF‐labeled electrochemical biosensor for the detection of Adenosine Triphosphate (ATP) was constructed using polyethylenimine‐reduced graphene oxide/Gold nanowire nanocomposite (PEI‐rGO/AuNWs) and exonuclease I (Exo I) assisted target recycling strategy. PEI‐rGO/AuNWs nanocomposite not only favor the immobilization of c‐DNA but also facilitate the electron transfer and elevate the electrode surface area. In addition, hierarchically porous Zr‐MOFs (HP‐UiO‐66), high stability and large mesoporous property, could load more signal molecules. In the presence of ATP, ATP aptamer was released from the c‐DNA, thus introducing MB@Zr‐MOF‐s‐DNA onto the electrode surface. Meanwhile, Exo I selectively digested the aptamer which bound with ATP, the released ATP participated new binding with the rest aptamer. Therefore, a significant increase in MB current intensity was observed. Under optimal conditions, the proposed aptasensor showed excellent performance with a linear range from 10−6 μM to 10 μM and a detection limit of 3.76×10−8 μM by differential pulse voltammetry. In addition, the proposed biosensor showed an outstanding performance with reproducibility, stability and anti‐interference ability, and was applied to the determination of ATP in real samples.