The invention provides a composite microsphere marker for conducting fluorescent marking on rear earth and a preparation method of the composite microsphere marker and relates to the technical field of functional polymer biomaterials. The preparation method includes the steps that polyvinylpyrrolidone, potassium persulfate / persulphate and styrene are added to an ethyl alcohol medium or a water medium to be dissolved and then placed into an ultrasonic-wave reactor, a polymerization reaction is generated under protection of nitrogen gas, and polystyrene nanometer microsphere dispersion liquid is obtained; then rare earth fluorescence complex 4, 7-dichloro sulfo benzene-1, 10 phenanthroline-2, 9 dicarboxylic acid-europium or 4, 4-di (1, 1, 1, 2, 2, 3, 3, -heptafluoride-4,6,-adipic ketone group) chlorine sulfo-adjacent-terphenyl- europium is fixed to the surface of a silicon dioxide microsphere in a chemical bonding mode, and finally the composite microsphere marker for conducting fluorescent marking on rear earth is prepared. The composite microsphere marker has the advantages of being long in fluorescent lifetime, good in stability, easy to synthesize, low in cost and easy to mark.