Ni-based catalysts supported on a support with 3D-mesopores, including Ni/KIT-6(EG), Ni/KIT-6(PS) and Ni/KIT-6(DS), were prepared by adding ethylene glycol, direct synthesis and post synthesis methods, respectively, and their catalytic properties were investigated for CO methanation as one of the core technologies of synthetic natural gas production in a continuous flow fixed-bed reactor. The catalysts were characterized by N2 adsorption-desorption, X-ray diffraction (XRD), transmission electron microscope (TEM), energy-dispersive X-ray spectroscopy (EDS), hydrogen temperature-programmed reduction (H2-TPR), hydrogen temperature-programmed desorption (H2-TPD) and thermal gravimetric analysis (TGA), respectively. The results showed that Ni/KIT-6(EG) exhibited the best catalytic performance with CO conversion of almost 100% and CH4 yield of 75% at 450 °C, atmospheric pressure and 60,000 mL/g/h due to the higher dispersion of Ni species, stronger reducibility of NiO and formation of smaller Ni nanoparticles fixed into 3D-mesopores, indicating that adding ethylene glycol was effective to improve catalytic performance of Ni-based catalyst for CO methanation. Moreover, compared with Ni/Al2O3(EG) prepared using Al2O3 as a support, Ni/KIT- 6(EG) showed better catalytic performance owing to the higher specific surface area, stronger reducibility of NiO and confinement effect of 3D-mesopores promoting to produce more active sites. After 60h lifetime test of Ni/KIT-6(EG) at 500 °C, atmospheric pressure and 60,000mL/g/h, 3D-mesopores were still maintained and no obvious agglomeration of Ni nanoparticles was observed, meaning that Ni species were still well dispersed into 3D-mesopores. As a consequence, Ni/KIT-6(EG) exhibited superior catalytic performance and stability, which makes it a promising candidate for CO methanation.