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Grain boundary enriched CuO nanobundle was reported to significantly increased the glucose diffusion as well as reduce the glucose oxidation barrier, thus displayed a lower onset potential of 94 mV, an extremely high sensitivity of 7474 μA mM−1 cm−2 in glucose detection and a high peak power density of 242 W m−2 in a glucose fuel cell. [Display omitted] • CuO nanobundle with high density and homogenous GBs was obtained by a facile and large-scale method. • A high sensitivity of 7474 μA mM−1 cm−2 in glucose detection and a high peak power density of 242 W m−2 in a glucose fuel cell are achieved. • CuO-NB possesses the faster glucose mass transfer due to the terraces in GBs dislocation surface. • Electrons accumulate on GBs interfaces promote glucose adsorption and decrease free energy of dehydrogenation step. Glucose oxidation reaction (GOR) plays a significant role in glucose fuel cells anode and glucose sensors. Therefore, optimizing the GOR catalyst nanostructure is auxiliary to their efficient operation. In this study, we present a cascade-assembled strategy to prepare CuO nanobundles (CuO-NB) with high-density and homogenous grain boundaries (GBs). The essence of activity in GOR that depended on GBs are thoroughly investigated. The increased glucose diffusion coefficient of CuO-NB means that GBs has a faster glucose mass transfer, which is attributed to the terraces in GBs dislocation surface. Furthermore, the accumulation of electrons on GBs makes the glucose adsorption increased and the free energy of dehydrogenation step decreased, leading to a lower glucose oxidation barrier. Therefore, CuO-NB is appropriate for non-invasive glucose detection and glucose fuel cells. This study sheds new light on the GBs effect in GOR and paves the way for developing high-efficiency electrocatalysts. [ABSTRACT FROM AUTHOR]