Graphdiyne is a new carbon nanomaterial after graphene. It has excellent electrical properties and can be chemically modified. Here, a graphdiyne oxide-polyurethane nanocomposite (GDYO-PU) was synthesized by chemical coupling and characterized with Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM) and scanning electron microscopy (SEM). A GDYO-PU modified glassy carbon electrode (GCE) was applied as a non-enzymatic glucose electrode. The electrochemical property of the GDYO-PU electrode was investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and amperometry in the presence of [Fe(CN)6]3−/4− in 0.1 M NaOH alkaline solution and at the bias potential of 0.2 V. Fast electro-deposition was observed for the electrochemical deposition of Fe(CN)6 3−/4− on the GDYO-PU electrode surface. Under the optimized conditions, the calibration curve of the nano electrode is linear over the concentration range of 0.2 to 6.4 μM, with high sensitivity of 377 μAmM−1cm−2 as well as a low detection limit (LOD) of 0.1 μM (S/N = 3). The GDYO-PU nanocomposite based non-enzyme glucose electrode is therefore promising for analysis of tiny volume of biological samples, which meets the requirement of minimal invasive measurement in clinic and family care.