Monomethoxy poly(ethylene glycol) Amine (mPEG5k-EDA) with the relative molecular mass of 5000 was prepared by two-step reaction. Monomethoxy poly(ethylene glycol) tosylate (mPEG5k-OTs) was synthesized by monomethoxy poly(ethylene glycol) 5000 (mPEG5k) and p-toluenesulfonyl chloride (p-TsCl) at first, and then reacted with ethylenediamine as nucleophile to obtain the target product. The optimal reaction conditions were obtained as follows by the orthogonal experiment: mole ratio between mPEG5k-OTs and ethylenediamine was 1:25, reaction temperature was 80℃ and reaction time was 24 h. The structures of product and intermediate were characterized by IR and 1H-NMR, and the variation of molecular weight of the product was detected by SDS-PAGE iodine staining method. The results showed that mPEG5k-EDA was prepared quickly and easily by this method, and the overall yield of the target product could be as high as 76.7% under the optimal experimental conditions. The purity of the target product was proved by SDS-PAGE without any crosslinking by-products.