The invention provides a method for breeding AA-high-producing strains by utilizing protoplast fusion and combining with fusant screening through fluorescence staining. In order to obtain mortierella alpina strains with high yield of arachidonic acid, the invention provides the method adopting the following scheme: two mortierella alpina strains with different superior characteristics are subjected to fusion treatment by adopting a protoplast fusion method, and fusants are screened by combining with the fluorescence staining method. The method provided by the invention comprises the following specific steps: culturing two parental strains, preparing protoplasts of the two parental strains, carrying out fluorescence staining on the protoplasts of the two parental strains, fusing the protoplasts of the two parental strains, and screening and verifying the fusants. The method provided by the invention is easy to operate, the process is easy to realize, and the breeding success probability is high. The method can be used for improving the yielding level of arachidonic acid producing strains, optimizing the fat components of the arachidonic acid product, and improving the characters of the strains.