Glucose detection is essential in clinical medicine, and the reasonable design of metal oxide electrocatalysts plays a crucial role in developing efficient nonenzymatic glucose (NEG) sensors. Herein, grain boundary/doping/architecture engineering is used to tailor the structures of CuO nanomaterials and tune their surface/electron-transfer properties toward enhanced electrocatalytic oxidation of glucose. Hierarchical N-doped CuO microflowers (N-CuO-MF) are synthesized using a facile hydrothermal method, followed by calcination. N-CuO-MF consist of ultrathin nanoflakes (ca. 20 nm), endowing them with a large specific surface area. Moreover, the nanoflakes are composed of ultrasmall nanoparticles, resulting in abundant grain boundaries. Notably, N-CuO-MF are derived from a precursor of Cu-based metal-organic framework (Cu-MOF) architectures, which is fabricated through a bottom-up route using glycerol as the capping agent/solvent and 1-hexadecyl-3-methylimidazolium bromide ([C