The arachidonic acid oil and its microbial fermentation production method relate to the microbial fermentation production method, overcome the shortcomings of low arachidonic acid content and high cost of products, and enhance production stability and food safety. The arachidonic acid oil is produced by Mortierella alpina M0223, the content of arachidonic acid residues is 65.5-73.5%, and the content of eicosapentaenoic acid residues is 0.69-3.7% of the content of arachidonic acid residues. %. The steps of the method are as follows: culturing and activating strains, culturing seeds, inserting strains into shake flasks or fermentation tanks for fermentation and culturing, collecting wet cells, drying and extracting oil. The invention greatly improves the product quality, reduces the cost, and avoids the problems of production stability and food safety caused by artificially modified strains. The refined oil of the present invention can be used as nutrition enhancer for infants and young children, food additive and raw material of medicine and cosmetics.