The invention relates to a standard sample preparation method for a synchronous radiation confocal fluorescence experiment. The method comprises the steps that S101 a copper film is deposited on the entire top surface of a silicon substrate, and the silicon substrate with the copper film is cut; S102 the copper film and the silicon substrate with partial thickness are etched to form a copper stripand a first substrate part, wherein the silicon substrate is adjacent to the copper film; and S103 the copper strip and the first substrate part are etched to form a copper strap and a second substrate part. A standard sample prepared by the invention is convenient for more accurate calibration of devices in the synchronous radiation confocal fluorescence experiment, and the accuracy of confocalmicro-element calibration is improved.