Ferroferric oxide (Fe3O4) nanospheres have been synthesized via a facile solvothermal procedure to serve as an electrode material for high performance non-enzymatic glucose sensor. The as-synthesized Fe3O4 nanospheres with a uniform size from 16 to 18 nm, which can increase the reaction contact area and the active sites in the process of glucose detection. Benefiting from the particular nanoscale structure, the Fe3O4 nanospheres obviously enhanced the activity of electrocatalytic oxidation towards glucose. When the Fe3O4 nanospheres material was used for non-enzymatic glucose sensor, several electrochemical properties including the high sensitivity 6560 μA mM−1 cm−2 (0.1–1.1 mM), limit of detection 33 μM (S/N = 3) and good long-term stability were well demonstrated. Furthermore, Fe3O4 nanospheres electrode confirmed the excellent performance of selectivity in glucose detection with the interfering substances existed such as urea, citric acid, ascorbic acid, and NaCl. Due to the excellent electrocatalytic activity in alkaline solution, the Fe3O4 nanospheres material can be considered as a promising candidate in blood glucose monitoring.