The invention discloses a preparation method of monodisperse cross-linked polymethyl methacrylate microspheres. The method comprises the following steps: enabling polymethyl methacrylate to react with an initiator, so as to obtain a solution A; enabling the polymethyl methacrylate to react with a cross-linking agent, so as to obtain a solution B; enabling the solution A to react with the solution B, and then collecting the monodisperse cross-linked polymethyl methacrylate microspheres in a reaction product. Compared with the prior art, the method has the advantages of being high in crosslinking density, continuous to react, simple to operate, short in reaction time, high in yield and the like. Compared with a common seed two-step swelling method for preparing the crosslinked microspheres, the seed microspheres do not need to be firstly synthetized; swelling for a plurality of times, and reacting for a plurality of times are not needed; a plurality of assistants are not added, so that the production efficiency can be greatly improved by two-step dispersion polymerization, and the method has great practicability.