Mesoporous NiMo-Al2O3 catalysts were synthesized without the structure-directing agent by a single-pot method and were studied for quinoline hydrodenitrogenation (HDN). These catalysts were characterized by N-2 physisorption, X-ray diffraction (XRD), temperature-programmed reduction by hydrogen (H-2-TPR), and UV-vis/diffuse reflection spectroscopy (UV-vis DRS). The Mo sulfidation and morphology structures of (Ni)MoS2 crystallites of sulfided catalysts were analyzed by X-ray photoelectron spectroscopy (XPS) and high-resolution transmission electron microscopy (HRTEM). The NiMo catalysts with different textural properties and metal-support interaction could be controlled by adjusting the hydrolysis process. The catalyst prepared with direct hydrolysis (the NM-dh sample) exhibited the best catalytic performance, with quinoline conversion of 96.9% and the DN products selectivity of 60.5%. The catalytic results highlighted the effect of the metal-support interaction on HDN performance. The incorporation of citric acid as a chelate additive resulted in a decrease in catalytic activity and selectivity, providing evidence that the catalytic HDN performance was very selective to the agglomeration of Mo species.