Non-enzymatic glucose sensors are attracting significant attention owing to their low cost, storage convenience and reusability; however, their poor anti-interference caused by their weak selectivity towards glucose limits their practicability. In the present study, a MIL-53(NiFe) metal-organic framework (MOF) was prepared on Ni foam to serve as a self-supported electrode for non-enzymatic glucose detection. Due to the abundant active sites in the MIL-53(NiFe) MOF and its good stability in an alkaline solution, the sensors exhibited a high sensitivity (41.95 mA mM-1 cm-2) and a low detection limit (0.67 μM). Moreover, the molecular sieve effect of the MIL-53(NiFe) MOF led to a remarkable anti-interference ability, even at the interference concentrations of up to 20% glucose, a higher value than that in human serum. In addition, a heat treatment was carried out to remove the residual terephthalic acid in the MOF tunnels, and this promoted the detection linear range to 2-1600 μM. The reusability, reproducibility and long-term stability of the sensors were also studied, and the results implied good practicability of the MIL-53(NiFe) MOF-based sensors. Furthermore, the good practicability of the sensors was verified by testing human serum samples. The results showed the relative standard deviation of 2.73% from the hospital results, and the standard recovery was nearly 100%.