Improved method for the measurement of substance comprising simultaneously measuring a plurality of specimens of a substance to be measured by enzyme immunoassay. The process comprises:a. reacting specimens and a material which will be specifically combined with the substance to be measured and which has been made in the solid phase;b. separating the reaction mixture in step (a) into the solid phase and the liquid phase;c. reacting the solid phase with an enzyme-labelled product of a material which will be specifically combined with the substance to be measured;d. separating the reaction mixture in the step (c) into the solid phase and the liquid phase;e. reacting the solid phase or the liquid phase with a substrate for the enzyme in the step (c); andf. measuring the optical density of the reaction mixture. The improvement comprises, in step (a), successively contacting the respective specimens with the material which will be specifically combined with the substance to be measured and which has been made in the solid phase, under cooling condition at a temperature ranging from 0.degree. C. to 15.degree. C., followed by simultaneously incubating them at a temperature ranging from room temperature to 45.degree. C. The fluctuation of results can be remarkably smaller by the employment of cooling means.