The invention relates to a method for preparing a micron monodisperse polymeric microsphere under a room temperature condition. According to the invention, the monodisperse polymeric microsphere is prepared by utilizing the reaction of mercaptan and isocyanate through precipitation polymerization without surface active agent or stabilizer. The method comprises the following steps: uniformly mixing mercaptan monomer with isocyanate monomer; under an effect of a basic catalyst, standing by and reacting for 1-8 hours under room temperature; washing the acquired product with a solvent; centrifugally precipitating; drying, thereby acquiring the monodisperse polymeric microsphere. The preparation method provided by the invention is simple and reliable; the reaction is performed under the room temperature; no sealing or stirring is required; the time consumption is short; the monomer dosage is large; the microsphere yield is high.