The development of high-efficient technologies for cancer biomarkers detection has attracted tremendous research effort for its great clinic significance. In this work, we designed a new type of flexible and robust nanohybrid microelectrode by modifying carbon fiber with dual nanoenzyme, i.e., AuPd alloy nanoparticles (AuPd-ANPs) decorated graphene quantum dots (GQDs) assembly, and explored its practical application in electrochemical sensing system for sensitive detection of cancer biomarker hydrogen peroxide (HO) in human breast cancer cells and tissue. For the preparation of dual nanoenzyme modified microelectrode, ionic liquid was used as the electrolyte for the effective electrodeposition of GQDs on carbon fiber substrate to form a close-packed assembly under a very negative potential, then the highly dense AuPd-ANPs were uniformly decorated on GQDs assembly by electrodeposition. In virtue of the structural merits and synergistic contribution of dual nanoenzyme in enhancing the electrocatalytic activity to HO, the resultant nanohybrid microelectrode exhibited good sensing performances for electrochemical detection of HO, including a high sensitivity of 371 μA cm mM, a wide linear range from 1.0 μM to 18.44 mM, a low detection limit of 500 nM (a signal-to-noise ratio of 3:1), as well as good selectivity and biocompatibility, which could be used for real-time tracking HO released from different types of human breast cells and in situ sensitive detection of HO in clinical breast cancer tissue.