In this work, highly monodispersed palladium-nickel (Pd-Ni) nanoparticles supported on reduced graphene oxide (rGO) were synthesized by the microwave-assisted methodology. The synthesized nanoparticles were used for modification of a glassy carbon electrode (GCE) to produce our final product as PdNi@rGO/GCE, which were utilized for non-enzymatic detecting of glucose. In the present study, electrochemical impedance spectroscopy (EIS), chronoamperometry (CA) and, cyclic voltammetry (CV) methods were implemented to investigate the sensing performance of the developed glucose electrode. The modified electrode, PdNi@rGO/GCE, exhibited very noticeable results with a linear working range of 0.05-1.1 mM. Moreover, an ultralow detection limit of 0.15 μM was achieved. According to the results of amperometric signals of the electrodes, no significant change was observed, even after 250 h of operation period. In addition, the highly monodisperse PdNi@rGO/GCE was utilized to electrochemical detection of glucose in real serum samples. In light of the results, PdNi@rGO/GCE has shown an excellent sensing performance and can be used successfully in serum samples for glucose detection and it is suitable for practical and clinical applications.