A method of preparing an integral multilayer analytical element such as for analysis of calcium comprising a water-impermeable light-transmissive support, a reagent layer containing a water-soluble indicator capable of reacting with an analyte to produce an optically detectable change, and a porous spreading layer containing a spreading action controller and/or an acid capable of decomposing the calcium compounds in a sample, superposed in this order, wherein the spreading action controller and/or the acid is dissolved in an organic solvent which does not dissolve the above water-soluble indicator, this solution is incorporated into the porous spreading layer, and this superposed material is dried. In the method of the present invention, the migration of the water-soluble indicator in the reagent layer at the time of incorporating the spreading action controller or the acid into the spreading layer is inhibited by using a particular organic solvent. Migrations of the indicator and colored material to the spreading layer are also lowered by the spreading action controller, and by these, analytical accuracy of the analytical element is improved. In the case of the analytical element for analysis of calcium, permeation of the acid into other layers than the spreading layer is inhibited, and permeation of a pH buffer in other layer(s) into the spreading layer is also inhibited by using a particular organic solvent. As a result, effective decomposition of calcium compounds in a sample and effective coloration can be achieved.