The invention relates to a method for preparing a coating with anti-reflection and anti-fog functions on the surface of an optical substrate based on the layer-by-layer assembly technology, belonging to the technical field of film coatings. The method comprises the following steps: firstly, introducing amino, carboxyl or hydroxyl on the surface of the substrate; then, alternately putting the substrate into a solution of polycation building basic elements and a solution of mesoporous silica nanoparticles for respectively soaking for 2-10min, and removing the physically adsorbed building basic elements after being soaked each time on the surface of the substrate by a soaking or washing method, thereby completing the preparation of a layer-by-layer assembly film of one cycle; and repeating the steps to prepare a layer-by-layer assembly film coating with controllable thickness on the substrate by using the layer-by-layer assembly technology. The method is suitable for substrates of plane surfaces, curved surfaces and irregular surfaces, such as eyeglass lenses of polycarbonate, swimming glasses and the like. The method not only can possibly enable the anti-reflection and anti-fog coating to be widely used in daily life but also can possibly enable the anti-reflection and anti-fog coating to be more widely used in other fields.