In this study, a series of Ni/ZnO-Al2O3 adsorbents were synthesized by a one-pot cation-anion double hydrolysis (CADH) method. The materials were characterized by N2 sorption, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), ultraviolet-visible diffuse reflectance spectroscopy (UV-vis), Raman spectroscopy, and H2 temperature-programmed reduction (H2-TPR). The reactive adsorption desulfurization (RADS) performance of the adsorbents was evaluated in a fixed bed reactor using thiophene in n-octane as a model fuel. Results showed that the adsorbents exhibited better RADS performance than those prepared using the conventional kneading method. The thiophene conversion and sulfur capacity of adsorbents decreased with increasing the crystallization temperature. Among all tested adsorbents, the Ni/ZnO-Al2O3 sample prepared at 28 °C presented the largest adsorption capacity and highest RADS reactivity. Textual characterization results indicated that the sample Ni/ZnO-Al2O3(28 °C) possessed relatively bigger pore size and larger pore volume than other samples, which may alleviate the pore shrinkage/blockage during the RADS process. A combination of XRD, UV-vis, and H2-TPR characterization results demonstrate that a high crystallization temperature favors the growth of inactive ZnAl2O4 crystals and induce the formation of more less-reducible Ni2+ ion, causing the loss of active ZnO phase and Ni0 atoms, which may be the reason for the lower RADS activity of the adsorbent synthesized at higher crystallization temperatures.