Nickel-iron hydrotalcites (NiFe-LDH) with various [Ni]/[Fe] ratios were prepared using co-precipitation method (NiFe-LDH CP) and hydrothermal method (NiFe-LDH HT), respectively. The crystal structure and chemical composition of NiFe-LDHs were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetry and differential scanning calorimetry (TGA-DSC) and scanning electron microscopy, and NiFe-LDHs were occupied as catalysts to establish heterogeneous Fenton systems for the degradation of methylene blue (MB). The catalytic potential of the catalysts was investigated through cyclic voltammetry analysis. The effects of the dosage of catalyst, initial solution pH and the amount of hydrogen peroxide on the removal of MB were investigated. The results showed that the optimum ratio of [Ni]/[Fe] in the preparation of NiFe-LDHs was 3. NiFe-LDH HT is much smaller and in uniform particle size, with better redox reversible characteristic and catalytic potential. The optimum conditions for the removal of MB catalyzed by NiFe-LDH CP and NiFe-LDH HT were both determined to be 0.15 g/L catalyst, 0.88 mmol/L hydrogen peroxide at pH 2, under which the chemical oxygen demand (COD) removal were 58.96% and 67.87%, respectively. The maximum apparent generation efficiency of ·OH were 46.21% and 49.24%, and NiFe-LDH CP and NiFe-LDH HT were verified to be of high stability.