In proteomics, glycosylation is the enzymatic process that attaches glycans to protein and produces glycoprotein. As an enzyme for non-specific digestion, pronase E is commonly used in the digestion of glycoprotein, with the advatanges of maintaining class and composition information of the digested glycan and identifying the glycosylation sites. A new magnetic nanosphere material, Fe3O4@NH2@CHO, is synthesized through Borch reduction reaction, to immobilize the non-specific pronase E onto the surface of the magnetic nanospheres, thereby reducing pronase E’s  self-digestion effects that can affect the analysis of glycan in MS. As a result, this method can largely reduce the complexity of the experimental system, which allows for greater sensitivity in MS measurements of the glycan samples. The synthesized material is characterized by Transmission Electron Microscopy, Zeta Electric Potential, Thermogravimetric Analysis, and Infra-Red Spectroscopy, and it has been proven that the desired material was successfully synthesized. Also, its immobilization efficiency and the immobilized pronase’s activity were evaluated. Experiments suggested that its immobilization amount has reached 365ug pronase E/mg Fe3O4@NH2@CHO ; the immobilized pronase has shown great activity.