In this study, ultrafine and uniform cuprous oxide (Cu₂O) nanocubes with different sizes and rough surfaces were prepared via a seed-mediated process. The Cu₂O nanocubes were formed by the self-assembly of the subsequently generated Cu₂O crystallites on the surface of the introduced seed medium. The obtained samples were used for non-enzymatic glucose detection and exhibited good capabilities. The Cu₂O nanocubes obtained from a beaker 3 modified electrode showed the best performance, such as high sensitivity (3213 μA mM⁻¹ cm⁻²), low limit of detection (LOD, 0.14 μM), acceptable linear range (0.05 to 5.15 mM) and fast electrocatalytic response (<1 s). In addition, the acceptable selectivity and long-term stability prove that Cu₂O nanocube modified non-enzyme glucose biosensors are promising for practical applications.