Abstract: A process for preparing and separating a monodisperse polyethylene glycol, including: dissolving a starting material of polyethylene glycol into an organic solvent for reaction with a compound represented by general formula I under catalytic action of an alkaline substance, to prepare a mixture of derivatives of polyethylene glycol having different polarities; separating the mixture of derivatives of polyethylene glycol by means of silica gel column chromatography, to obtain a monodisperse derivative of polyethylene glycol; and hydrolyzing the derivative of polyethylene glycol to obtain a monodisperse polyethylene glycol. The starting material is industrially prepared polyethylene glycol with a wide source and low cost, which provides a guarantee for popularization and application of a monodisperse polyethylene glycol product.