Abstract: A flexible graphite sheet can be used as a self-supported substrate material for sensors owing to its excellent conductivity and flexibility. Copper nanoparticle/flexible graphite sheet self-supporting enzyme-free glucose sensors were prepared by a hydrothermal method using copper sulfate as the elemental copper source and ascorbic acid as the reducing agent. Results indicate that both the molar ratio of copper sulfate to ascorbic acid and the hydrothermal temperature affect the microstructure and sensor performance. The optimal sensor obtained with a ratio of 1:0.5 at 150℃ has a low detection limit of 1.05 μmol/L, and high sensitivities of 7 254.1 μA·mM^-1·cm^-2(R²=0.996 1)from 0.1 to 3.4 μmol/L and 3 804.5 μA·mM^-1·cm^-2 (R²=0.999 5) from 3.4 to 5.6 μmol/L. The sensor possesses excellent anti-interference properties against sodium chloride, acetaminophenol, ascorbic acid, dopamine, and uric acid, and has good reproducibility.