The invention provides preparation of a high-effective aspergillus flavus (Aspergillus sp.Y2K) chitosan endonuclease mutant. The mutation enzyme can be used for hydrolyzing chitosan to produce oligosaccharide or monosaccharide. The method mainly includes the following steps: 1) cloning a DNA sequence of a matured recombinant chitosanase gene, with mutation at two loci A286G and C287T, onto an expression vector pPICZ[alpha]A of pichia pastoris; and 2) integrating the recombinant plasmid to pichia pastoris host bacteria X-33 through an electric-conversion process to form the recombinant chitosanase mutant which can high-effectively secrete and express and high-effectively degrade chitosan and is high in purity. Compared with a non-mutation enzyme, the chitosan endonuclease mutant is increased in activity by three times. The chitosan endonuclease is widely applied to the fields of foods, medicines, bio-resources, etc.