Currently, real time monitoring of chemical substances in vivo and in vitro has gained enormous attraction, and many researches reports have been focused on the design and construction of high-performance biosensor devices. In this work, a high-performance sensor was constructed by taking advantage of the excellent electrochemical activity and high-index facets of Au-Pd nanocubes and the large surface of rGO. Glassy carbon electrodes (GCEs) were modified by both Au-Pd nanocubes and rGO nanocomposites via physical adsorption. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were utilized to characterize and identify this unique nanostructure. These three-dimensional nanocomposites possess a high electroactive surface area and an excellent electrical conductivity, which resulted in favorable electroreduction activity toward H2O2 with a lower detection limit of 4 nM, a wide linear range from 0.005 μM to 3.5 mM and a rapid response time. Furthermore, the proposed sensor exhibited desirable performance in the detection of endogenous H2O2 in human serum samples and real-time monitoring of H2O2 released from living breast cancer cell lines. In summary, this work not only provides a potential method to construct a physiological and pathological H2O2 biosensor but also makes a valuable contribution to the early diagnosis of different cancers.