The development of novel catalyst with high catalytic activity is important for electrochemical non-enzymatic glucose sensing. Here, iridium single-atom/nickel oxide nanoparticle/N-doped graphene nanosheet (Ir1/NiO/NG) with the loading of 1.13 wt% Ir was successfully synthesized for constructing electrochemical non-enzymatic glucose sensor for the first time. The morphology and structure of Ir1/NiO/NG were characterized by XRD, SEM, TEM, HRTEM, and XPS, and the presence of Ir SAs was confirmed by AC-HAADF-STEM. The Ir1/NiO/NG shows 65 mV lower oxidation potential and 3.3 times higher response current than Ni(OH)2/NG. In addition, Ir1/NiO/NG exhibits high sensitivity (70.09 μA mM-1 cm-2), excellent selectivity, low detection limit (2.00 μM), and great stability (91.53% current remaining after 21 days) for electrochemical non-enzymatic glucose sensing. The outstanding catalytic and sensing performance of Ir1/NiO/NG is mainly attributed to synergistic effect of Ir SAs, NiO nanoparticles, and highly conductive NG, which modulate the electronic and geometric structure of Ir1/NiO/NG. This work shows the promising potential of SACs in electrochemical sensing.