The invention discloses an immobilized protease and an application method of the immobilized protease in preparation of milk casein peptides. The main preparation method of the immobilized protease is as follows: firstly, immobilized carrier chitosan microspheres are prepared; secondly, the chitosan microspheres are added into protease solution; after oscillation and absorption, glutaraldehyde solution is dripped into the solution; after cross linking, the immobilized protease is obtained. The method for preparation of the milk casein peptides adopts the immobilized protease to hydrolyze the milk caseins. The immobilized protease can be reused for a plurality of times, thereby the use efficiency is high and the production cost is effectively reduced; moreover, the immobilized protease hasthe advantages of convenient product separation, low cost, mild production conditions and easy control of the hydrolysis degree. The milk casein peptide product prepared has the advantages of high peptide content, superior peptide molecule weight distribution range, good quality, no bitterness, high safety and so on. The content (according to the dry basis) of the milk casein peptides prepared bythe method is as high as 75.05 percent by detection; the peptide molecule weight distribution range is that: the molecular weight which is lower than 2409 daltons reaches 97.79 percent.